Dual function molecules for histone deacetylase inhibition and ataxia telangiectasia mutated activation and methods of use thereof

ABSTRACT

Dual function compounds are provided that may be inhibitors of histone deacetylase (HDAC) and activators of ataxia telangiectasia mutated (ATM). Pharmaceutical compositions and methods of use are also provided that utilize such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Patent application Ser. No.14/636,736, filed on Mar. 3, 2015, the entirety of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates generally to compounds that inhibithistone deacetylase (HDAC) and activate ataxia telangiectasia mutated(ATM) and more particularly, but not exclusively, to dual functioncompounds that may inhibit HDAC and activate ATM and pharmaceuticalcompositions and methods of treating diseases that may beneficiallyutilize such compounds.

BACKGROUND OF THE INVENTION

A variety of diseases are known in the field to elude common treatmentmethods. For example, certain diseases and disorders that implicate thehistone deacetylase (HDAC) proteins have continued to evade knowntherapeutics and treatment methodologies.

Accordingly, a need exists in the field for compounds, compositions, andmethods for treating such elusive diseases and disorders, includingcertain cancers and neurological disorders.

SUMMARY OF THE INVENTION

The present invention meets the needs in the field by providing dualfunction compounds that may inhibit HDAC and activate ATM and may beused in the treatment of certain cancers, neurological disorders, andimmunological disorders. Indeed, the compounds of the invention may beused in pharmaceutical compositions and methods of treatment incombating these and other related diseases.

In a first aspect, the invention includes a compound, such as a dualfunction, compound having the formula:

wherein R¹, R³, R⁷, and R⁸ may be independently selected from the groupconsisting of H, hydroxy, halogen and, optionally substituted, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, amino,alkoxy, carboxy, carbalkoxy, carboxamido, sulfonyl, sulfinyl,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosufonyl,dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkylsulfonylalkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl, anddialkylaminosulfinylalkyl.

R² and R⁹ may be independently selected from the group consisting of Hand, optionally substituted, sulfinyl, sulfonyl, alkyl, alkenyl,cycloalkyl, aryl, heterocycle, and heteroaryl.

R⁴ may be selected from the group consisting of H and optionallysubstituted alkyl.

R⁵ may be selected from the group consisting of H, and optionallysubstituted alkyl and indole.

R⁶ may be selected from the group consisting of H and optionallysubstituted alkyl.

X may be selected from the group consisting of:

wherein R¹⁰ may be selected from the group consisting of H and,optionally substituted, alkyl, alkenyl, cycloalkyl, aryl, heterocycle,and heteroaryl. In preferred aspects, when X is maleimide orN-carbonylmaleimide, n+r=0.

n may be 0 or 1; r may be an integer from 0 to 3; q may be an integerfrom 3 to 10; the dashed line may indicate the presence of a single bondor a double bond as allowed; with the proviso that, where X is asubstituent other than maleimide or N-carbonylmaleimide and R⁵ is asubstituent other than indole, then R³ is a substituent selected fromthe group consisting of hydroxy, halogen and, optionally substituted,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl,amino, alkoxy, carboxy, carbalkoxy, carboxamido, sulfonyl, sulfinyl,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosufonyl,dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkylsulfonylalkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl, anddialkylaminosulfinylalkyl, with the dashed line indicating the presenceof a double bond; and the pharmaceutically acceptable salts of thecompound of Formula I.

In preferred embodiments, q may be an integer from 4 to 6. For example,q may be 5. Moreover, in certain embodiments of Formula I, where X is asubstituent other than maleimide or N-carbonylmaleimide and R⁵ is asubstituent other than indole, then R³ is 2-alkyl or 3-alkyl.

In one embodiment, the compound of Formula I may be a compound selectedfrom the group consisting of:

and the pharmaceutically acceptable salts thereof.

In a further embodiment, the compound of Formula I may be a compoundhaving the formula:

wherein R¹¹, R¹³, R¹⁴, and R¹⁶ may be independently selected from thegroup consisting of H, hydroxyl, halogen and, optionally substituted,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl,amino, alkoxy, carboxy, carbalkoxy, carboxamido, sulfonyl, sulfinyl,monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosufonyl,dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy,alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl,alkylsulfonylalkyl, monoalkylaminosulfonylalkyl,dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl, anddialkylaminosulfinylalkyl.

X may be selected from the group consisting of:

wherein R¹⁸ may be selected from the group consisting of H and,optionally substituted, alkyl, alkenyl, cycloalkyl, aryl, heterocycle,and heteroaryl.

R¹² and R¹⁵ may be independently selected from the group consisting of Hand, optionally substituted, alkyl, sulfinyl, and sulfonyl.

R¹⁷ may be selected from the group consisting of H and optionallysubstituted alkyl.

n may be an integer from 3 to 10; the dashed line may indicate thepresence of a single bond or a double bond as allowed; with the provisothat, where X is amide, then R¹³ is 2-alkyl or 3-alkyl, the dashed lineindicating the presence of a double bond; and the pharmaceuticallyacceptable salts of the compound of Formula II. In certain embodiments,n may be an integer from 4 to 6. For example, n may be 5.

Additionally, the compound of Formula I or II may be a compound selectedfrom the group consisting of:

and the pharmaceutically acceptable salts thereof.

In another embodiment, the compound of Formula I may be a compoundhaving the formula:

wherein R¹⁹ and R²¹ may be independently selected from the groupconsisting of H, hydroxyl, halogen and, optionally substituted alkyl,aryl, heterocycle, heteroaryl, sulfonyl, sulfinyl, alkoxy, and amino.

X may be selected from the group consisting of:

wherein R²³ may be selected from the group consisting of H and,optionally substituted, alkyl, alkenyl, cycloalkyl, aryl, heterocycle,and heteroaryl.

R²⁰ may be selected from the group consisting of H and, optionallysubstituted, alkyl, sulfinyl, and sulfonyl.

R²² may be selected from the group consisting of H and optionallysubstituted alkyl.

r may be an integer from 0 to 4; q may be an integer from 3 to 10; thedashed line may indicate the presence of a single bond or a double bondas allowed; with the proviso that, R²¹ is 2-alkyl or 3-alkyl with dashedline indicating the presence of a double bond; and the pharmaceuticallyacceptable salts of the compound of Formula III. In certain embodiments,q may be an integer from 4 to 6. For example, q may be 5.

In another embodiment, the compound of Formula I or III may be acompound selected from the group consisting of:

and the pharmaceutically acceptable salts thereof.

In another embodiment, the compound of Formula I may be a compoundhaving the formula:

wherein R²⁴ and R²⁶ may be independently selected from the groupconsisting of H, hydroxyl, halogen and, optionally substituted alkyl,aryl, heterocycle, heteroaryl, sulfonyl, sulfinyl, alkoxy, and amino.

R²⁵ may be selected from the group consisting of H and, optionallysubstituted, alkyl, sulfinyl, and sulfonyl.

R²⁷ may be selected from the group consisting of H and optionallysubstituted alkyl; m may be an integer from 3 to 10; the dashed line mayindicate the presence of a single bond or a double bond as allowed; withthe proviso that, R²⁶ is a substituent selected from the groupconsisting of hydroxyl, halogen and, optionally substituted alkyl, aryl,heterocycle, heteroaryl, sulfonyl, sulfinyl, alkoxy, and amino, with thedashed line indicating the presence of a double bond and thepharmaceutically acceptable salts of the compound of Formula IV. Incertain embodiments, m may be an integer from 4 to 6. For example, m maybe 5.

In another embodiment, the compound of Formula I or IV may be a compoundselected from the group consisting of:

and the pharmaceutically acceptable salts thereof.

In another aspect, the invention includes a pharmaceutical formulationthat may be in unit dosage form. The pharmaceutical formulation of theinvention may include a compound of Formula I and may be provided in anamount effective to inhibit histone deacetylase (HDAC) and activateataxia telangiectasia mutated (ATM) in a patient in need thereof and mayinclude at least one physiologically compatible carrier medium.

The pharmaceutical formulation of the invention may include a compoundof Formula II, Formula III, and/or Formula IV.

In an additional aspect, the invention includes a method of treating adisease in a patient in need thereof. The method may includeadministering a therapeutically effective amount of at least onecompound configured to inhibit histone deacetylase (HDAC) and activateataxia telangiectasia (ATM). The at least one compound may be a compoundof Formula I.

In one embodiment, the method may include administering at least onecompound selected from the group consisting of:

and the pharmaceutically acceptable salts thereof.

In other embodiments, the methods of the invention may includeadministering at least one compound of Formula II, Formula III, and/orFormula IV. The methods of the invention may include the administrationof the at least one compound in dosage unit form that may furtherinclude a physiologically acceptable carrier medium.

In further embodiments, the diseases treated by the methods of theinvention may include a disease selected from the group consisting ofcancer, immunological disorders, and neurological disorders.

When the disease treated by the methods of the invention is cancer, thecancer may be selected from those cancers listed in Table 1. In certainaspects, the cancer may be selected from the group consisting of gastriccancer, prostate cancer, colon cancer, breast cancer, Non-Hodgkin'slymphoma, ovarian cancer, sarcoma, lung cancer, leukemia, myeloma,testicular cancer, cervical cancer, pancreatic cancer, head and neckcancer, rectal cancer, and brain cancer. The method may further includethe step of administering to said patient an amount of radiotherapyconfigured to treat said cancer.

When the disease treated by the methods of the invention is animmunological disorder, the immunological disorder may be selected fromthe group consisting of systemic lupus and erythematosus rheumatoidarthritis.

When the disease treated by the methods of the invention is aneurological disorder, the neurological disorder may be selected fromthe group consisting of stroke, Huntington's disease, spinal muscularatrophy (SMA), Parkinson's disease, Alzheimer's, Multiple Sclerosis, andAmyotrophic Lateral Sclerosis (ALS). In preferred aspects, theneurological disorder treated by the methods of the invention may beAlzheimer's disease or multiple sclerosis.

In still further embodiments, the method of the invention may be asecond or third line method of treatment for the patient andadministration of the compound occurs after performance of a first orsecond therapy on the patient that failed to treat the disease.

In a further aspect, the invention includes a method of treatment thatmay include sensitizing cancerous cells to radiotherapy and protectingnon-cancerous cells from radiotherapy in a patient in need thereof,wherein cancerous cells are sensitized to radiotherapy by inhibitinghistone deacetylase (HDAC) and non-cancerous cells are protected fromradiotherapy by activating ataxia telangiectasia mutated (ATM). Themethod may include administering a therapeutically effective amount ofat least one compound of Formula I.

In other embodiments, the method may include administering at least onecompound of Formula II, Formula III, and/or Formula IV.

In still further embodiments, the cancerous cells may be the result of acancer selected from those cancers listed in Table 1. For example, thecancerous cells may be the result of a cancer selected from the groupconsisting of gastric cancer, prostate cancer, colon cancer, breastcancer, Non-Hodgkin's lymphoma, ovarian cancer, sarcoma, lung cancer,leukemia, myeloma, testicular cancer, cervical cancer, pancreaticcancer, head and neck cancer, rectal cancer, and brain cancer.

The method of the invention may further include the step ofadministering to the patient an amount of radiotherapy configured totreat the cancerous cells.

Accordingly, as briefly described herein, the present invention includescompounds, compositions, and methods of treatment that provide treatmentsolutions to answer the needs in the field.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary and the following detailed description of theexemplary embodiments of the present invention may be further understoodwhen read in conjunction with the appended drawings, in which:

FIG. 1 schematically illustrates exemplary embodiments of Formula I.

FIG. 2 schematically illustrates certain selected embodiments of FormulaI.

FIGS. 3A and 3B demonstrate the activity ofN-(6-(carboxy)-6-oxohexyl)-1H-indole-2-carboxamide (SP-1-105) as anactivator of ATM. The activity data is demonstrated in tabular form(FIG. 3A) and graphical form (FIG. 3B). The ATM activity was determinedby examining the fold change in phospho-ATM in MCF7 cells.

FIGS. 4A and 4B demonstrate the activity ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161) as an activator of ATM. The activity data is demonstrated intabular form (FIG. 4A) and graphical form (FIG. 4B). The ATM activitywas determined by examining the fold change in phospho-ATM in MCF7cells.

FIGS. 5A and 5B demonstrate the activity ofN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide (SP-1-163)as an activator of ATM. The activity data is demonstrated in tabularform (FIG. 5A) and graphical form (FIG. 5B). The ATM activity wasdetermined by examining the fold change in phospho-ATM in MCF7 cells.

FIGS. 6A and 6B demonstrate the activity of(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide(SP-1-169) as an activator of ATM. The activity data is demonstrated intabular form (FIG. 6A) and graphical form (FIG. 6B). The ATM activitywas determined by examining the fold change in phospho-ATM in MCF7cells.

FIGS. 7A and 7B demonstrate the activity of(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(SP-1-171) as an activator of ATM. The activity data is demonstrated intabular form (FIG. 7A) and graphical form (FIG. 7B). The ATM activitywas determined by examining the fold change in phospho-ATM in MCF7cells.

FIGS. 8A and 8B graphically illustrate the activity of certain exemplarycompounds of the invention as HDAC inhibitors. Specifically, thecompounds tested included:N-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161) (FIG. 8A);N¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide (SP-1-163)(FIG. 8A);(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide(SP-1-169) (FIG. 8B); and(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(SP-1-171) (FIG. 8B).

FIG. 9 demonstrates, in tabular form, the cytotoxic effect of certaincompounds of the invention on breast cancer cells (MCF7 cells) andhealthy breast tissue cells (184A1 cells) in both an MTT Cytotoxicityassay and Clonogenic Cytotoxicity assay. Specifically, the followingcompounds were tested:N-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161); N¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide(SP-1-163);(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide(SP-1-169); and(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(SP-1-171).

FIG. 10 demonstrates, in tabular form, the effects ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161) on the radiation survival curves and parameters for normalbreast epithelial cells (184A1 cells) and breast cancer cells (MCF7cells).

FIGS. 11A and 11B graphically illustrate the cytotoxic effect ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161), DIM, and DMSO against normal breast epithelial cells (184A1cells) (FIG. 11A) and breast cancer cells (MCF7 cells) (FIG. 11B).

FIG. 12 graphically illustrates the level of ATM phosphorylation inducedafter exposure to various indole compounds at 1 μM as a percentage ofthe ATM phosphorylation level of cells treated with ionizing radiationat 6 Gy. The phospho-ATM levels were measured at 30 minutes, 1 hr, 2 hr,4 hr, and 6 hr post exposure to radiation. Specifically, indole,3-methyl indole, and 2,3-dimethyl indole were compared. 2,3-dimethylindole demonstrated a significant increase in ATM phosphorylation whencompared to indole and 3-methyl indole. MCF7 cells were treated and thenuclear fraction was extruded and analyzed via Elisa assay for P-ATM(S1982).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to compounds, and compositionsthat include such compounds, which may be HDAC inhibitors and ATMactivators. More specifically, the compounds of the invention are dualfunction compounds as represented in Formulas I-IV, which may be used intreating diseases that implicate HDAC and/or ATM, such as certaincancers, immunological diseases, and neurological diseases.

Regarding the compounds of the invention, which are encompassed withinFormulas I-IV, as used herein, the term “alkyl” denotes branched orunbranched hydrocarbon chains, having about 1 to 10 carbons, such as,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, 2-methylpentyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl and the like. “Substituted alkyl”includes an alkyl group optionally substituted with one or morefunctional groups which are attached commonly to such chains, such as,hydroxy, halogen, mercapto or thio, cyano, alkylthio, carboxy,carbalkoxy, amino, nitro, alkoxy, or optionally substituted, alkenyl,alkynyl, heterocyclyl, aryl, heteroaryl, and the like to form alkylgroups such as trifluoro methyl, 3-hydroxyhexyl, 2-carboxypropyl,2-fluoroethyl, carboxymethyl, cyanobutyl, phenethyl, benzyl and thelike.

The term “halogen” or “halo” as used herein alone or as part of anothergroup refers to chlorine, bromine, fluorine, and iodine.

The term “alkoxy” refers to alkyl-O—, in which alkyl is as definedabove.

The term “alkylthio” refers to alkyl-S—, in which alkyl is as definedabove.

The term “alkylamino” refers to alkyl-N—, in which alkyl is as definedabove.

The term “carboxy” refers to the moiety —C(═O)OH.

The term “carbalkoxy” refers to the moiety —C(═O)O-alkyl, in which alkylis as defined above.

The term “carboxamido” refers to the moiety —C(═O)—NR′R″, in which R′and R″, each may independently represent H, alkyl, or aryl, all asdefined herein.

The term “alkylcarbonylamino” refers to the moiety —NR′C(═O)—R″, inwhich R′ and R″, each may independently represent H, alkyl, or aryl, allas defined herein.

The term “alkylsulfonyl” refers to the moiety —S(═O)₂-alkyl, in whichalkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)₂-alkyl, whereinalkyl is as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfinyl” refers to themoiety —S(═O)NR′R″ in which R′ and R″ each may independently representH, alkyl, or aryl, all as defined herein.

The term “amino(monoalkylamino-, dialkylamino-)sulfonyl” refers to themoiety —S(═O)₂NR′R″, in which R′ and R″ each may independently representH, alkyl, or aryl, all as defined herein.

The term “alkylsulfonylamino” refers to the moiety —NHS(═O)₂-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyloxy” refers to the moiety —OS(═O)₂OH.

The term “alkoyxsulfonyloxy” refers to the moiety —OS(═O)₂O-alkyl, inwhich alkyl is as previously defined.

The term “alkylsulfonyloxy” refers to the moiety —OS(═O)₂-alkyl, inwhich alkyl is as previously defined.

The term “hydroxysulfonyl” refers to the moiety —S(═O)₂OH.

The term “alkoxysulfonyl” refers to the moiety —S(═O)₂O-alkyl, whereinalkyl is as previously defined.

The term “alkylsulfonylalkyl” refers to the moiety -alkyl-S(═O)₂-alkyl,wherein each alkyl may be as previously defined.

The term “amino(monoalkylamino-, dialkylamino-)sulfonylalkyl” refers tothe moieties -alkyl-S(═O)₂—NR′R″, wherein alkyl is as previouslydefined, and R′ and R″ each may independently represent H, alkyl, oraryl, all as defined herein.

The term “amino(monoalkylamino-, dialkylamino-)sulfinylalkyl” refer tothe moieties -alkyl-S(═O)—NR′R″, wherein alkyl is as previously defined,and R″ and R″ each may independently represent H, alkyl, or aryl, all asdefined herein.

Unless otherwise indicated, the term “cycloalkyl” as employed hereinalone or as part of another group includes saturated or partiallyunsaturated (containing 1 or more double bonds) cyclic hydrocarbongroups containing 1 to 3 rings, including monocyclicalkyl, bicyclicalkyland tricyclicalkyl, containing a total of 3 to 20 carbons forming therings, preferably 3 to 10 carbons, forming the ring and which may befused to 1 or 2 aromatic rings as described for aryl, which includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl, cyclododecyl, and cyclohexenyl.

“Substituted cycloalkyl” includes a cycloalkyl group optionallysubstituted with 1 or more substituents such as halogen, alkyl,substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl, aryloxy,cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino,amino, nitro, cyano, thiol and/or alkylthio and/or any of thesubstituents included in the definition of “substituted alkyl.”

Unless otherwise indicated, the term “alkenyl” as used herein by itselfor as part of another group refers to straight or branched chain of 2 to20 carbons, preferably 2 to 12 carbons, and more preferably 2 to 8carbons in the normal chain, which include one or more double bonds inthe normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-butenyl,4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl,4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl,4,8,12-tetradecatrienyl, and the like. “Substituted alkenyl” includes analkenyl group optionally substituted with one or more substituents, suchas the substituents included above in the definition of “substitutedalkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “alkynyl” as used herein by itselfor as part of another group refers to straight or branched chain of 2 to20 carbons, preferably 2 to 12 carbons and more preferably 2 to 8carbons in the normal chain, which include one or more triple bonds inthe normal chain, such as 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl,3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl,3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl, 4-dodecynyl and the like.“Substituted alkynyl” includes an alkynyl group optionally substitutedwith one or more substituents, such as the substituents included abovein the definition of “substituted alkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “aryl” or “Ar” as employed hereinalone or as part of another group refers to monocyclic, bicyclic, and/orpolycyclic aromatic groups containing 6 to 10 carbons in the ringportion (such as phenyl or naphthyl including 1-naphthyl and 2-naphthyl)and may optionally include one to three additional rings fused to acarbocyclic ring or a heterocyclic ring, such as aryl, cycloalkyl,heteroaryl, or cycloheteroalkyl rings or substituted forms thereof.

“Substituted aryl” includes an aryl group optionally substituted withone or more functional groups, such as halo, alkyl, haloalkyl (e.g.,trifluoromethyl), alkoxy, haloalkoxy (e.g., difluoromethoxy), alkenyl,alkynyl, cycloalkyl-alkyl, cycloheteroalkyl, cycloheteroalkylalkyl,aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy,alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, arylalkenyl,aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl,heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro,cyano, amino, substituted amino wherein the amino includes 1 or 2substituents (which are optionally substituted alkyl, aryl or any of theother substituents recited herein), thiol, alkylthio, arylthio,heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylaminocarbonyl,arylaminocarbonyl, aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy,alkylcarbonylamino, arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl,arylsulfonylamino, or arylsulfonaminocarbonyl and/or any of the alkylsubstituents recited herein.

Unless otherwise indicated, the term “heteroaryl” as used herein aloneor as part of another group refers to a 5- to 7-membered aromatic ringwhich includes 1, 2, 3 or 4 hetero atoms such as nitrogen, oxygen orsulfur and such rings fused to an aryl, cycloalkyl, heteroaryl orheterocycloalkyl ring (e.g. benzothiophenyl, indolyl), and includespossible N-oxides. “Substituted heteroaryl” includes a heteroaryl groupoptionally substituted with 1 to 4 substituents, such as thesubstituents included above in the definition of “substituted alkyl” and“substituted cycloalkyl.” Substituted heteroaryl also includes fusedheteroaryl groups which include, for example, quinoline, isoquinoline,indole, isoindole, carbazole, acridine, benzimidazole, benzofuran,isobenzofuran, benzothiophene, phenanthroline, purine, and the like.

Moreover, the terms “heterocyclo,” “heterocycle,” or “heterocyclicring,” as used herein, refer to an unsubstituted or substituted stable5- to 7-membered monocyclic ring system which may be saturated orunsaturated, and which consists of carbon atoms and from one to fourheteroatoms selected from N, O or S, and wherein the nitrogen and sulfurheteroatoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heterocyclic ring may be attached at anyheteroatom or carbon atom which results in the creation of a stablestructure. Examples of such heterocyclic groups include, but are notlimited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl,oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl,thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,oxazolidinyl, isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,thiazolidinyl, isothiazolyl, thiadiazolyl, tetrahydropyranyl,thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, andoxadiazolyl.

As used herein, the term “optionally substituted” may indicate that achemical moiety referred to, for example, alkyl, aryl, heteroaryl, maybe unsubstituted or substituted with one or more groups including,without limitation, alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl,aryl, heterocycle, heteroaryl, hydroxyl, amino, alkoxy, halogen,carboxy, carbalkoxy, carboxamido, monoalkylaminosulfinyl,dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl,alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy,alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl,monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl,monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl and the like. Thechemical moieties of Formulas I-IV, above, that may be optionallysubstituted include alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl,aryl, heterocycle, and heteroaryl. For example, optionally substitutedalkyl may include both propyl and 2-chloro-propyl. Additionally,“optionally substituted” is also inclusive of embodiments where thenamed substituent or substituents have multiple substituents rather thansimply a single substituent. For example, optionally substituted arylmay include both phenyl and 3-methyl-5-ethyl-6-chloro-phenyl.

The compounds of the invention may be administered as salts, which arealso within the scope of this invention. Pharmaceutically acceptable(i.e., non-toxic, physiologically compatible) salts are preferred. Ifthe compounds of the invention have, for example, at least one basiccenter, they can form acid addition salts. These are formed, forexample, with strong inorganic acids, such as mineral acids, for examplesulfuric acid, phosphoric acid or a hydrohalic acid, with strong organiccarboxylic acids, such as alkane carboxylic acids of 1 to 4 carbon atomswhich are unsubstituted or substituted, for example, by halogen, forexample acetic acid, such as saturated or unsaturated dicarboxylicacids, for example oxalic, malonic, succinic, maleic, fumaric, phthalicor terephthalic acid, such as hydroxycarboxylic acids, for exampleascorbic, glycolic, lactic, malic, tartaric or citric acid, such asamino acids, (for example aspartic or glutamic acid or lysine orarginine), or benzoic acid, or with organic sulfonic acids, such as(C₁-C₄) alkyl or arylsulfonic acids which are unsubstituted orsubstituted, for example by halogen, for example methyl- orpara-toluene-sulfonic acid. Corresponding acid addition salts can alsobe formed having plural basic centers, if desired.

The compounds of the invention having at least one acid group (e.g.,carboxylic acid or hydroxamic acid) can also form salts with suitablebases. Representative examples of such salts include metal salts, suchas alkali metal or alkaline earth metal salts, for example sodium,potassium or magnesium salts, or salts with ammonia or an organic amine,such as morpholine, thiomorpholine, piperidine, pyrrolidine, a mono, dior tri-lower alkylamine, for example ethyl, tert-butyl, diethyl,diisopropyl, triethyl, tributyl or dimethyl-propylamine, or a mono, dior trihydroxy lower alkylamine, for example mono, di or triethanolamine.Corresponding internal salts may also be formed.

For example, certain salts of the compounds described herein whichcontain a basic group include monohydrochloride, hydrogensulfate,methanesulfonate, phosphate or nitrate. Moreover, certain salts of thecompounds described herein which contain an acid group include sodium,potassium and magnesium salts and pharmaceutically acceptable organicamines.

All stereoisomers of the compounds of the invention, either in a mixtureor in pure or substantially pure form, are considered to be within thescope of this invention. The compounds of the invention may haveasymmetric centers at any of the carbon atoms including any one of thesubstituents. Consequently, compounds of the invention may exist inenantiomeric or diastereomeric forms or in mixtures thereof.Furthermore, where a stereocenter existing in a compound of theinvention is represented as a racemate, it is understood that thestereocenter may encompass the racemic mixture of R and S isomers, the Sisomers, and the R isomers. The processes for preparation of suchcompounds can utilize racemates, enantiomers, or diastereomers asstarting materials. When diastereomeric or enantiomeric products areprepared, they can be separated by conventional methods including,chromatographic, chiral HPLC, fractional crystallization, ordistillation. Some compounds of the present invention have groupsincluding alkenyls, iminyls, and the like, which may exist as entgegen(E) or zusammen (Z) conformations, in which case all geometric formsthereof, both E and Z, cis and trans, and mixtures thereof, are withinthe scope of the present invention. Accordingly, when such geometricisomeric products are prepared, they can be separated by conventionalmethods for example, chromatographic, HPLC, distillation orcrystallization.

Specific compounds of the invention include those compounds set forth inFIG. 1. In certain aspects, the compounds of the invention include thosecompounds set forth in FIG. 2. Certain preferred compounds of theinvention includeN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide (i.e.,SP-1-161) and N¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)octanediamide(i.e., SP-1-163).

The compounds of the invention may be used as part of a therapy ormethodology in treating a variety of diseases or conditions thatimplicate HDAC inhibition and/or ATM activation. For example, suchdiseases may include cancer, immunological disorders, and neurologicaldisorders.

Cancer is the second leading cause of death in the United States afterheart disease. The American Cancer Society estimates that 1,665,540 newcancer cases are expected to have been diagnosed in 2014 with 585,720cancer-related deaths.

The standard treatments of cancer include surgery, radiotherapy, andchemotherapy. Each treatment modality carries risks and benefits, andcancer recurrences underlie efforts to improve the outcomes oftreatment. In particular, recent advances in surgical and radiationtherapy technologies, employing computational and robotic methods, haveplateaued efficacy of local-regional treatments. Moreover, targetedagents to personalize chemotherapy have altered the cancer treatmentparadigm.

Radiation therapy (i.e., radiotherapy) involves the treatment of cancerand other diseases using ionizing radiation. Ionizing radiation depositsenergy that injures or destroys cells in targeted tissues by damagingtheir genetic material and subsequently interfering with a cell'sability to grow and/or replicate. Radiation exposure damages cancercells and normal cells, but the normal cells activate processes tobetter repair themselves and may continue to function properly.Radiotherapy may be used to treat solid tumors (e.g., cancers of thehead and neck, breast, prostate, rectum, uterus, lung, brain, kidney,uterus, and cervix). Radiotherapy may also be used to treat cancers suchas leukemias and lymphomas. Radiotherapies used for leukemias andlymphomas may include total body radiation therapy in protocolspreparing patients for bone marrow transplants. Radiotherapy may be moreeffective when the targeted cancer tissues are more sensitive to theeffects of radiation than surrounding normal tissues.

The radiation responses of different cancers or tumors may vary as afunction of histology, cellular doubling time, oxygenation, nutrientavailability, repair capacity, and other factors. Some cancers arereadily cured using ionizing radiation doses within normal tissuetolerances, while other types of cancer may not be very responsive toradiation. Furthermore, radiation responses of tumors with the samehistology may show considerable heterogeneity and reduce the therapeuticeffects of the therapy. Thus, a primary challenge facing radiotherapy isthe differentiation between the more radiosensitive tumors versus lessradiosensitive tumors and the surrounding healthy tissues.

Investigations into the molecular bases underlying cellular radiationresponses have provided dramatic mechanistic insight. Signaltransduction pathways have been implicated to play important roles incellular responses to ionizing radiation. Induction of gene expressionby these cascades under various conditions has been shown to result incell cycle arrest, activation of DNA repair processes, and activation ofprogrammed cell death (apoptosis). Disruption of critical signalingpathways in cancer cells may result in enhanced cytotoxic effectsfollowing radiation exposure. Certain cells may be disrupted byinterfering with the histone acetylation and deacetylation processes ofthe cells.

Histone acetylation and deacetylation play important roles in chromatinfolding and maintenance. Acetylation appears to play a role in theepigenetic regulation of chromatin structure, and gene expression,through the balance of histone acetyltransferase (HAT) and histonedeacetylase (HDAC) activities. Increased acetylation of histones leadsto changes in chromatin structure and accessibility for key cellularproteins to specific target sites. HATs acetylate lysine groups at theamino terminal tails of nuclear histones to neutralize positive chargeson the histones, yielding a more open, transcriptionally activechromatin structure. In contrast, the HDACs deacetylate and suppresstranscription. In this model, inhibitors of HDACs bias the balancetoward a more acetylated state. Such a shift in the relative activitiesof these enzymes may affect gene expression necessary for DNA repair,replication, cell cycle checkpoint activation and tumor suppression.

Human HDACs may be divided into four classes based on structure,sequence homology, and domain organization. Class I consists of HDACs 1,2, 3, 8, and 11, albeit a recent report puts HDAC 11 into a new class,class IV, based on a phylogenetic analysis. Class I HDACs are nuclearand play roles in cell proliferation and apoptosis. Class II includesHDACs 4, 5, 6, 7, 9, and 10. These enzymes are characterized by a largeNH₂-terminal domain or a second catalytic site and their expression ismore restricted, suggesting roles in cellular differentiation anddevelopment. Class III enzymes, include the sirtuins (SIRTs), and areNAD-dependent deacetylases. These are not inhibited by Trichostatin A(TSA) or other hydroxamates.

HDACs are found in the nuclear and cytoplasmic compartments. Althoughthey are involved in critical cellular functions, such as cell cycleregulation and apoptosis, a key function of HDACs is transcriptionalregulation. HDACs function as components of large multi-proteincomplexes that bind to promoters and repress transcription. Class IIcompounds shuttle between the nucleus and the cytoplasm. However,certain classes of HDACs have conserved deacetylase core domains ofapproximately 400 amino acids and zinc binding sites. It is the coredomain that presents the principal target for design of inhibitory smallmolecules.

In response to DNA damage, signal transduction pathways may be activatedto regulate cell cycle arrest, repair, differentiation, apoptosis, andtranscription. Such responses are a complex feature of the cellularradiation phenotype, and their effectiveness may determine cell survivalor death. DNA damage checkpoints generate signals that arrest cell cycleprogression until the damage is repaired. When damaged DNA is repaired,checkpoint signals are reversed to resume cell cycle progression. SuchDNA-directed processes are accompanied by highly localized changes inchromatin structure. Various recent studies have implicated chromatinstructure in DNA damage signaling and repair. Post-translational histonemodifications regulate chromatin structure and access for proteins todamaged DNA sites as reported for repair and signaling proteins to thedamaged regions of DNA.

Early HDAC inhibitors (e.g., benzamides) were investigated asdifferentiating agents, without full understanding of their molecularmechanisms. Some of these agents have advanced to clinical trials. Thefull recognition of the potential for HDAC inhibitors was advanced withthe discovery and development of hydroxamic acid inhibitors. Hydroxamicacid based compounds (e.g., suberoylanilide hydroxamic acid (SAHA)) havebeen developed for clinical application, and have proven to berelatively non-toxic. SAHA has been approved by the FDA for thetreatment of cutaneous T-cell lymphoma. Certain HDAC inhibitors havebeen described in U.S. Pat. Nos. 7,507,828; 7,842,835; 8,067,600;8,222,451; and 8,748,463; the entirety of which are incorporated hereinby reference.

Other chemical families of HDAC inhibitors, including depsipeptide andvalproic acid, have been shown to inhibit cancer cell growth in vitroand in vivo. Modulation of p53, ErbB1, ErbB2 and Raf-1 expression havebeen observed following exposure of lung cancer cells to depsipeptide, adrug currently in clinical trials. For example, Valproic acid has beenused clinically as an anti-epileptic agent, with excellent reasonabletoxicity profile and has been shown to be involved in the proteolysis ofHDAC 2.

Several lines of evidence support targeting HDACs to achieve radiationsensitization of cancer cells following exposures to HDAC inhibitors.The responses of cells to ionizing radiation may be viewed as a complexphenotype involving various signal transduction pathways associated withthe activation of stress responses, cell cycle regulation, DNA repairand regulation of apoptosis.

Damage sensing and repair proteins, including ATM, MRE11, γ-H2AX and53BP1, have been associated with changes in chromatin structure.Proteins that bind directly to ends of broken DNA include Ku, DNAPK andPARP. ATM kinase is considered a primary regulator of responses to DNAdouble strand breaks and activates a number of downstream effectors,including H2AX, MDC1/NFBD1, 53BP1, Brca1, and MRN (Mre11, Rad50 andNbs1). These various molecules provide potential intermediate endpointsfor studies of effects of HDAC inhibitors on radiation sensitivities ofcancer cells.

Regarding ATM in particular, ATM may mediate the cell repair responseafter DNA damage (e.g., double strand breaks (DSBs)) or during periodsof oxidative stress. ATM may be activated by its phosphorylation atserine 1981 (Ser1981) triggered by ionizing radiation induced DNAdamage, leading to phosphorylation of critical factors involved in DNArepair, apoptosis, and cell cycle checkpoint regulation. ATM recruitmentto and activation by DSBs requires the MRN complex which functions bothupstream and downstream of ATM. MRN senses DSBs and activates ATM, butit is also phosphorylated and activated by ATM. MRN participates moredirectly in DNA repair by binding and tethering broken DNA ends close toone another and by processing DNA ends via the nuclease activity ofMre11. ATM may also be indirectly activated by Trichostatin A (TSA), anHDAC inhibitor, by a process that involves chromatin changes in theabsence of DNA breaks.

As used herein, the term “ATM activation” refers to the phosphorylationof ATM, which provides phospho-ATM. ATM may be directly or indirectlyactivated. For direct ATM activation, a ligand or compound may activateATM by a process that is not the downstream result of HDAC inhibition.In certain instances, HDAC inhibition may result in some measurable ATMactivation due to the resulting down stream effects of HDAC inhibition,which may include cell damage. However, without being limited to any onetheory, 3,3′-diindolylmethane (DIM) is a direct activator of ATM andprotects against γ radiation by stimulation of an ATM-driven DDR-likeresponse, without causing DNA damage. This response may involvesignalling through an MRN/ATM/BRCA1 pathway. By contrast, indirect ATMactivation arises where a ligand or compound activates ATM as abyproduct of the inhibition of HDAC protein.

Certain compounds of the invention (e.g., dual function compounds) aredirect ATM activators. Indeed, certain compounds of the invention mayprovide direct ATM activation activity, in addition to indirect ATMactivation activity which may result from HDAC inhibition. Theseactivities may be measured by examining the phosphorylation of ATM upontreatment with a compound of the invention. As will be discussed herein(see Example 13, FIG. 12), the compounds of the invention may includeboth a hydroxamic acid moiety and an indole moiety where at least theindole moiety is demonstrated to enhance direct ATM activation.Therefore, preferred compounds of the invention are dual functioncompounds in that they are HDAC inhibitors and ATM activators, which maydirectly activate ATM.

Certain chemical classes of HDAC inhibitors are radiation sensitizers.As used herein, the term “radiosensitizing agent” which may be read alsoas a “radiosensitizer” denotes an agent having an effect of enhancingthe sensitivity of cancerous and/or neoplastic cells to radiation. As ageneralization, chemosensitization and radiosensitization are importantproperties of HDAC inhibitors and may offer expanded clinicalopportunities for these agents. General properties that may be expectedto have an effect on radiation sensitivities of cancer cells includedifferentiation, growth inhibition, changes in gene expression andapoptosis. Key reported acetylation mechanisms have involved histonesand tubulin and a variety of other non-histone proteins.

Generally, chemotherapeutic compounds, such as HDAC inhibitors, may havevaried bioactivities. For example, chemotherapeutic compounds may havecytotoxic activity against cancerous cells and/or non-cancerous cells.Additionally, chemotherapeutic compounds may also exhibit additionalproperties such as the ability to sensitize cells, such as cancerouscells, to radiation. Alternatively, chemotherapeutic compounds may beradiation protectants that protect cells, such as non-cancerous cells,from the effects of radiation. Indeed, certain HDAC inhibitors mayinduce radiation sensitization in target tumor cells while normal cellsmay be more resistant and are relatively spared or protected from theeffects of radiation.

Therapeutic ratios may be determined by measuring the effects of drugson cancers and on normal tissues. Radiation toxicities to organs at riskmay affect normal tissues adjacent to the treated volume (such as rectumor bladder in the treatment of a pelvic tumor), or in sites receivingtransit dose (such as the pelvic bone marrow). Others have shownradiation protection of normal cells by HDAC inhibitors.

As described above, certain HDAC inhibitors may indirectly activate ATMand may be used as therapeutic agents to relax chromatin and hencesensitize cells to DNA-damaging drugs and/or radiation. Timelyactivation and inactivation of ATM are required for efficient repair,and any ATM perturbation may inhibit the ability of cells to resist DNAdamage.

Regarding ATM more specifically, ATM is a protein kinase mutated in thehuman disease ataxia telangiectasia (A-T). ATM has been a focus ofinvestigation because of the unusual radiosensitive phenotype of cellsfrom A-T patients. Because investigating ATM signalling has yieldedvaluable insights into the DNA damage response, redox signalling, andcancer, ATM has an important role in the repair of radiation-inducedDSBs of DNA and potentially of radiation protection of normal tissues.Indeed, ATM activation by DIM mitigates radiation injury in cells andanimals.

Accordingly, dual function compounds that inhibit HDAC and activate ATMare beneficial in that they may, for example, sensitize cancerous cellsto radiation while simultaneously aiding in the protection of healthycells and tissues about the cancerous tissues from such radiation. Theseproperties may be in addition to the dual function compound's cytotoxicactivity, which may be measured against both cancerous cells andnon-cancerous cells.

Regarding immunological diseases, the compounds of the invention may beused in methods of treating diseases that are the result of over-activeimmunity.

Regarding neurological diseases, millions of people worldwide enduresuch debilitating diseases that implicate HDAC proteins and may betreated by the compounds of the invention. Neurological diseases affecta vast number of humans of all ages. In the United States, over 500,000people each year experience a stroke, making it the third leading causeof death and the primary cause of disability. One in twenty people isafflicted with Alzheimer's disease by the age of 65, and almost 40percent of the population have the disease by age 80. More than 600,000people suffer from Parkinson's disease and over 200,000 from multiplesclerosis. Every year, greater than 10,000 people die from amyotrophiclateral sclerosis (ALS). The impact of neurological disease is not onlydevastating for the patients, but also for their families.

Although considerable effort has been invested in the design ofeffective therapies, neurological diseases continue to threaten theworldwide population and lessen their quality of life. The compounds ofthe invention may be used in compositions or methods for treating suchneurological disorders that implicate HDAC proteins. Specifically, thecompounds of the invention may be used in treating stroke, Huntington'sdisease, spinal muscular atrophy (SMA), Parkinson's disease,Alzheimer's, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis(ALS). In certain preferred aspects, the compounds of the invention maybe used in treating Alzheimer's disease and multiple sclerosis.

The present invention provides solutions for treating diseases byproviding compounds, compositions, and methods of treatment. When suchdiseases may include, but are not limited to, cancers and neurologicaldiseases.

As used herein, the terms “treat,” “treatment,” and/or “treating” mayrefer to the management of a disease, disorder, or pathologicalcondition (e.g., cancer, neoplastic disorder, immunological disorder, orneurological disorder) with the intent to cure, ameliorate, stabilize,prevent, or control the disease, disorder, or pathological condition.Regarding control of the disease, disorder, or pathological conditionmore specifically, “control” may include the absence of diseaseprogression, as assessed by the response to the methods recited herein,where such response may be complete (e.g., placing the disease inremission) or partial (e.g., slowing the spread of cancerous cells andtissues and/or preventing, slowing, or halting metastasis). The terms“treat,” “treatment,” and/or “treating” may further encompass, withrespect to the treatment of cancer, the sensitization of cancerous cellsand tissues (e.g., neoplastic cells and tissues) to radiation and/or theprotection of non-cancerous cells from the effects of radiation.

For example, a patient responding to the methods of treatment disclosedin the present invention may exhibit the absence of disease progression(e.g., halting the growth and/or spread of neoplastic cells and tissues)over another patient that does not receive the methods of treatmentdescribed herein.

Certain cancers that may be treated by the methods of the invention,with or without additional irradiation, are set forth in Table 1.

TABLE 1 Selected cancers that may be treated by the methods of theinvention. Exemplary Solid Tumors: acoustic neuroma adenocarcinomaangiosarcoma astrocytoma basal cell carcinoma bile duct carcinomabladder carcinoma breast cancer bronchogenic carcinoma cervical cancerchordoma choriocarcinoma colon cancer colorectal cancer craniopharygiomacystadenocarcinoma embryonal carcinoma endotheliosarcoma ependymomaepithelial carcinoma esophagaelcancer Ewing's tumor fibrosarcomaglioblastomamultiforme glioma hemangioblastoma hepatoma kidney cancerleiomyosarcoma liposarcoma lung cancer lymphangioendotheliosarcomalymphangiosarcoma medullary carcinoma medulloblastoma melanomameningioma mesothelioma myxosarcoma nasal cancer neuroblastomaoligodendroglioma oral cancer osteogenic sarcoma ovarian cancerpancreatic cancer papillary adenocarcinomas papillary carcinomapinealoma prostate cancer rabdomyosarcoma renal cell carcinomaretinoblastoma sebaceous gland carcinoma seminoma skin cancer squamouscell carcinoma stomach cancer sweat gland carcinoma synovioma testicularcancer small cell lung carcinoma throat cancer uterine cancer Wilms'tumor Exemplary Blood Cancers: acute erythroleukemic leukemia acutelymphoblastic B-cell leukemia acute lymphoblastic T-cell leukemia acutelypmhoblastic leukemia acute megakaryoblastic leukemia acute monoblasticleukemia acute myeloblastic leukemia acute myelomonocytic leukemia acutenonlymphocytic leukemia acute promyelocytic leukemia acuteundifferentiated leukemia chronic lymphocytic leukemia chronicmyelocytic leukemia hairy cell leukemia multiple myeloma ExemplaryLymphomas: heavy chain disease Hodgkin's disease multiple myelomanon-Hodgkin's lymphoma polycythemia vera Waldenstrom's macroglobulinemia

Certain specific cancers that may be treated by methods of the inventioninclude of gastric cancer, prostate cancer, colon cancer, breast cancer,Non-Hodgkin's lymphoma, ovarian cancer, sarcoma, lung cancer, leukemia,myeloma, testicular cancer, cervical cancer, pancreatic cancer, head andneck cancer, rectal cancer, and brain cancer. In certain aspects of theinvention, cancer treatment methods may include the application ofradiation as described herein.

The immunological disorders that may be treated by the methods of theinvention include systemic lupus and erythematosus rheumatoid arthritis.

The neurological disorders that may be treated by the methods of theinvention include stroke, Huntington's disease, spinal muscular atrophy(SMA), Parkinson's disease, Alzheimer's, Multiple Sclerosis, andAmyotrophic Lateral Sclerosis (ALS).

In determining the biological activity of the compounds of the inventionagainst HDAC and/or ATM or diseases that may be mediated by HDAC and/orATM (e.g., cancer) as they may be used in methods of the invention, thestructure of certain compounds may be compared to an HDAC and/or ATMpharmacophore. As used herein, the term “pharmacophore” refers to theensemble of steric and electronic features that are necessary to ensurethe optimal supramolecular interactions with a specific biologicaltarget structure (e.g., HDAC and/or ATM) and to trigger, activate,block, inhibit or modulate the biological target's biological activity,as the case may be. See, IUPAC, Pure and Applied Chemistry (1998) 70:1129-1143.

In comparing the biological activity of the compounds of the inventionagainst HDAC and/or ATM, biological activity may be correlated to thespecific structures of the compounds of the invention in the developmentof a pharmacophore model. As used herein, the term “pharmacophore model”refers to a representation of points in a defined coordinate systemwherein a point corresponds to a position or other characteristic of anatom or chemical moiety in a bound conformation of a ligand and/or aninteracting polypeptide, protein, or ordered water. An ordered water isan observable water in a model derived from structural determination ofa polypeptide or protein. A pharmacophore model can include, forexample, atoms of a bound conformation of a ligand, or portion thereof.A pharmacophore model can include both the bound conformations of aligand, or portion thereof, and one or more atoms that interact with theligand and are from a bound polypeptide or protein. Thus, in addition togeometric characteristics of a bound conformation of a ligand, apharmacophore model can indicate other characteristics including, forexample, charge or hydrophobicity of an atom or chemical moiety. Apharmacophore model can incorporate internal interactions within thebound conformation of a ligand or interactions between a boundconformation of a ligand and a polypeptide, protein, or other receptorincluding, for example, van der Waals interactions, hydrogen bonds,ionic bonds, and hydrophobic interactions. A pharmacophore model can bederived from 2 or more bound conformations of a ligand.

The compounds of the invention may be administered as described herein,or in a form from which the active agent can be derived, such as aprodrug. A “prodrug” is a derivative of a compound described herein, thepharmacologic action of which results from the conversion by chemical ormetabolic processes in vivo to the active compound. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues is covalentlyjoined through an amide or ester bond to a free amino, hydroxyl orcarboxylic acid group of Formulas I-IV. The amino acid residues includebut are not limited to the 20 naturally occurring amino acids commonlydesignated by one or three letter symbols but also include, for example,4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,3-methylhistidine, beta-alanine, gamma-aminobutyric acid, citrulline,homocysteine, homoserine, ornithine and methionine sulfone. Additionaltypes of prodrugs are also encompassed. For instance, free carboxylgroups can be derivatized as amides or alkyl esters. Prodrug esters asemployed herein includes esters and carbonates formed by reacting one ormore hydroxyls of compounds of the method of the invention with alkyl,alkoxy, or aryl substituted acylating agents employing procedures knownto those skilled in the art to generate acetates, pivalates,methylcarbonates, benzoates and the like. As further examples, freehydroxyl groups may be derivatized using groups including but notlimited to hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 115. Carbamate prodrugs of hydroxyl and amino groupsare also included, as are carbonate prodrugs, sulfonate prodrugs,sulfonate esters and sulfate esters of hydroxyl groups. Free amines canalso be derivatized to amides, sulfonamides or phosphonamides. All ofthe stated prodrug moieties may incorporate groups including but notlimited to ether, amine and carboxylic acid functionalities. Moreover,any compound that can be converted in vivo to provide the bioactiveagent (e.g., a compound of formula I) is a prodrug within the scope ofthe invention. Various forms of prodrugs are well known in the art. Acomprehensive description of prodrugs and prodrug derivatives aredescribed in: (a) The Practice of Medicinal Chemistry, Camille G.Wermuth et al., Ch 31, (Academic Press, 1996); (b) Design of Prodrugs,edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Designand Development, P. Krogsgaard-Larson and H. Bundgaard, eds., Ch. 5,pgs, 113-191 (Harwood Academic Publishers, 1991).

In general, prodrugs may be designed to improve the penetration of adrug across biological membranes in order to obtain improved drugabsorption, to prolong duration of action of a drug (slow release of theparent drug from a prodrug, decreased first-pass metabolism of thedrug), to target the drug action (e.g. organ or tumor-targeting,lymphocyte targeting), to modify or improve aqueous solubility of a drug(e.g., i.v. preparations and eyedrops), to improve topical drug delivery(e.g. dermal and ocular drug delivery), to improve thechemical/enzymatic stability of a drug, or to decrease off-target drugeffects, and more generally in order to improve the therapeutic efficacyof the compounds utilized in the invention.

A compound used in practicing any method of the invention may beadministered in an amount sufficient to induce the desired therapeuticeffect in the recipient thereof. Thus the term “therapeuticallyeffective amount” as used herein refers to an amount of a compound ofthe invention that is sufficient to treat a disease in accordance withthe invention by administration of one or more of the compounds offormulas I-IV or a prodrug thereof. Preferably, the therapeuticallyeffective amount refers to the amount appropriate to inhibit HDAC andactivate ATM in a patient. For example, the term therapeuticallyeffective amount may include the amount of a compound of the inventionnecessary to detectably sensitize cancerous cells to radiotherapy anddetectably protect non-cancerous cells from radiotherapy. In addition,the term therapeutically effective amount may include the amount of acompound necessary, for example, to bring about a detectabletherapeutic, preventative, or ameliorative effect in a patient having adisease as set forth herein. The effect may include, for example, thereduction, prevention, amelioration, or stabilization of symptoms orconditions associated with a disease as described herein.

For example, the therapeutically effective amount of a compound of theinvention that may sensitize cancerous or neoplastic cells to radiationmay be that amount that enhances the inhibitory or damaging effect ofradiation on cancer cells by at least 10%, at times by at least 20%,30%, 40%, 50%, 60%, 70% 80%, 90% and even at times by 99-100% of theinhibitory or damaging effect of the radiation on the cancer cells ascompared to the effect of radiation of the same cancerous and/orneoplastic cells, without sensitization.

The compounds and/or compositions of the invention that may sensitizecancerous or neoplastic cells to radiation may be administered in one ormore doses, at least a portion thereof being given to the patient priorto the patient's exposure to a radiation. When a treatment scheduleinvolves administration of several doses of the compound and/orcomposition, the doses may be the same or different, e.g. escalating orde-escalating amounts per administration. In addition, when referring toa radiosensitizing compound it should be understood as also encompassinga combination of such compounds.

The compounds and/or compositions of the invention are applicable fortreating disease in any mammal. Exemplary mammals included laboratoryanimals, including rodents such as mice, rats and guinea pigs; farmanimals such as cows, sheep, pigs and goats; pet animals such as dogsand cats; and primates such as monkeys, apes and humans. The compoundsused in the methods of the invention are preferably used in the humantreatments.

The methods of the invention may include irradiating a selected tissueof the patient before, during, and/or after a compound of the invention(or pharmaceutical composition containing such compound) that maysensitize cancerous or neoplastic cells to radiation and protecthealthy, non-cancerous cells and tissues from radiation has beenadministered to the patient. Regarding the application of radiation(“radiation therapy” or “radiotherapy”) to the patient or subject moregenerally, such therapy may encompass any ionizing radiation known tothose having ordinary skill in the art. Generally, radiation therapy,and in particular ionizing radiation includes applying to a selectedtissue, such as a selected tissue comprising cancerous and/or neoplasticcells, a dose of ionizing radiation or two or more fractions of ionizingradiation. The ionization radiation is defined as an irradiation dosewhich is determined according to the disease's characteristics at theselected tissue and therapeutic decision of a physician. The term“fractionated dose(s)” may include, for example, conventionalfractionation, hyperfractionation, hypofractionation, and acceleratedfractionation). The amount of radiation and doses thereof should besufficient to damage the highly proliferating cells' genetic material,making it impossible for the irradiated cells to continue growing anddividing.

In certain aspects, fractionated irradiation may vary from daily doses(e.g. one or more times per day) given for a period of weeks, or to onceweekly doses given for a period of weeks or months. Indeed, radiationmay be applied in dosages of about 0.1 Gy to about 100 Gy. For example,the dosage may be about 5 to 15 Gy.

In certain fractionated irradiation methods, irradiation dosing mayinclude the application of about 0.1 to about 20 Gy or from about 1 Gyto about 10 Gy or from about 1 Gy to about 3 Gy in a single session,which may be repeated several times over the course of about 1 to 10weeks, or preferably about 2 to 5 weeks. In certain embodiments of theinvention, the radiation dose may be about 30 to 60 Gy at 1 to 5 Gyfractions over a period of about 2 to 5 weeks.

In other exemplary aspects, three different fractionation schemes may beused in accordance with the invention.

In one embodiment, radiation doses from 1 Gy to 3 Gy in daily fractionsfor several weeks (e.g., about 2 to 8 weeks) to achieve cumulative dosesof about 20 Gy to 80 Gy.

In another embodiment, large fraction radiation therapy may includedoses of 4 Gy to 25 Gy. This fractionated irradiation scheme may includethe delivery of about 1 fraction to 5 fractions delivered over about 1-2weeks. This type of radiation may be referred to as stereotacticradiosurgery or stereotactic body radiation therapy.

In a further embodiment, brachytherapy may be used, which is deliveredusing low dose and rate techniques or high-dose rate techniques,typically delivering doses of about 4 Gy to 10 Gy per day with techniqueand fractionation specific to the clinical situation as would beunderstood by a person having ordinary skill in the art.

As set forth above, the compounds and/or compositions of the inventionmay be administered before, after, or together with the radiation. Onecycle of radiation therapy as well as several cycles of radiation ispossible, dependent on the reduction of tumor size or extent ofproliferation. Such sequences of radiosensitization treatments andionizing irradiation are repeated as needed to abate and, optimally,reduce or eliminate the spread of the cancer or neoplastic cells in thetissue or region of tissue that is selected for treatment. Accordingly,the total dose and the radiation regimen will depend, inter alia, on thecancer type, type of compound that results in radiosensitization,irradiated area, physical condition of the patient and many otherconsiderations appreciated by those having ordinary skill in the art.

In addition to the administration of a compound of the invention and theirradiation of the patient, the methods of the invention may include theadministration of a therapeutically effective amount of an additionalchemotherapeutic agent to the patient. The chemotherapeutic agent may beprovided before, during, or after at least one of the steps ofadministering the radiosensitizing agent and irradiating a selectedtissue of the patient. Therefore, the chemotherapeutic agent may beprovided at various points during the methods of the invention for thetreatment of disease. In certain aspects, the chemotherapeutic agent maybe administered concurrently with or after the step of irradiating theselected tissue of the patient.

The compound(s) described herein may also be administered at a dose inrange from about 0.01 mg to about 200 mg/kg of body weight per day. Adose of from 0.1 to 100, and preferably from 1 to 30 mg/kg per day inone or more applications per day should be effective to produce thedesired result. By way of example, a suitable dose for oraladministration would be in the range of 1-30 mg/kg of body weight perday, whereas a typical dose for intravenous administration would be inthe range of 1-10 mg/kg of body weight per day. In an exemplaryembodiment, the compounds of the invention may be administered at a doseof about 200 mg to 600 mg per day. For example, the compounds of theinvention may be administered at a dose of about 400 mg per day.

Of course, as those skilled in the art will appreciate, the dosageactually administered will depend upon the condition being treated, theage, health and weight of the recipient, the type of concurrenttreatment, if any, and the frequency of treatment. Moreover, theeffective dosage amount may be determined by one skilled in the art onthe basis of routine empirical activity testing to measure thebioactivity of the compound(s) in a bioassay, and thus establish theappropriate dosage to be administered.

The compounds used in certain methods of the invention may typically beadministered from 1-4 times a day, so as to deliver the above-mentioneddaily dosage. However, the exact regimen for administration of thecompounds described herein will necessarily be dependent on the needs ofthe individual subject being treated, the type of treatment administeredand the judgment of the attending medical specialist. As used herein,the term “subject” or “patient” includes both humans and animals.

In general, the compounds used in the methods of the invention can beadministered to provide radiosensitization as set forth above using anyacceptable route known in the art, either alone or in combination withone or more other therapeutic agents. Thus, the compound(s) of theinvention can be administered orally, parenterally, such as byintravenous or intraarterial infusion, intramuscular, intraperitoneal,intrathecal or subcutaneous injection, by liposome-mediated delivery,rectally, vaginally, by inhalation or insufflation, transdermally or byotic delivery.

The orally administered dosage unit may be in the form of tablets,caplets, dragees, pills, semisolids, soft or hard gelatin capsules,aqueous or oily solutions, emulsions, suspensions or syrups. Suitabledosage forms for parenteral administration include injectable solutionsor suspensions, suppositories, powder formulations, such asmicrocrystals or aerosol spray. The active agents of the invention mayalso be incorporated into a conventional transdermal delivery system.

As used herein, the expression “physiologically compatible carriermedium” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface agent agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants, fillers and the like as suited for the particular dosageform desired. Remington: The Science and Practice of Pharmacy, 20^(th)edition, A. R. Genaro et al., Part 5, Pharmaceutical Manufacturing, pp.669-1015 (Lippincott Williams & Wilkins, Baltimore, Md./Philadelphia,Pa.) (2000) discloses various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Except insofar as any conventional pharmaceutical carriermedium is incompatible with either the radiosensitizing orchemotherapeutic compounds used in the present invention, such as byproducing an undesirable biological effect or otherwise interacting inan deleterious manner with any other component(s) of a formulationcomprising such compounds or agents, its use is contemplated to bewithin the scope of this invention.

For the production of solid dosage forms, including hard and softcapsules, the agents of the invention may be mixed with pharmaceuticallyinert, inorganic or organic excipients, such as lactose, sucrose,glucose, gelatine, malt, silica gel, starch or derivatives thereof,talc, stearic acid or its salts, dried skim milk, vegetable, petroleum,animal or synthetic oils, wax, fat, polyols, and the like. For theproduction of liquid solutions, emulsions or suspensions or syrups onemay use excipients such as water, alcohols, aqueous saline, aqueousdextrose, polyols, glycerine, lipids, phospholipids, cyclodextrins,vegetable, petroleum, animal or synthetic oils. For suppositories onemay use excipients, such as vegetable, petroleum, animal or syntheticoils, wax, fat and polyols. For aerosol formulations, one may usecompressed gases suitable for this purpose, such as oxygen, nitrogen andcarbon dioxide. Pharmaceutical compositions or formulations may alsocontain one or more additives including, without limitation,preservatives, stabilizers, e.g., UV stabilizers, emulsifiers,sweeteners, salts to adjust the osmotic pressure, buffers, coatingmaterials and antioxidants.

The present invention further includes controlled-release,sustained-release, or extended-release therapeutic dosage forms foradministration of the compounds of the invention, which involvesincorporation of the compounds into a suitable delivery system in theformation of certain compositions. This dosage form controls release ofthe compound(s) in such a manner that an effective concentration of thecompound(s) in the bloodstream may be maintained over an extended periodof time, with the concentration in the blood remaining relativelyconstant, to improve therapeutic results and/or minimize side effects.Additionally, a controlled-release system would provide minimum peak totrough fluctuations in blood plasma levels of the compound.

In pharmaceutical compositions used in practicing the method of theinvention, the specified compound(s) may be present in an amount of atleast 0.5 and generally not more than 95% by weight, based on the totalweight of the composition, including carrier medium and/or supplementalactive agent(s), if any. Preferably, the proportion of compound(s)varies between 30-90% by weight of the composition.

The methods of the present invention will normally include medicalfollow-up to determine the therapeutic or prophylactic effect broughtabout in the subject undergoing treatment with the compound(s) and/orcomposition(s) described herein.

Specific compounds used in the compositions and methods of the inventioninclude those compounds set forth in FIG. 1. In certain aspects, thecompounds of the invention include those compounds set forth in FIG. 2.Certain preferred compounds of the invention includeN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide (i.e.,SP-1-161) and N¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)octanediamide(i.e., SP-1-163).

In additional aspects of the methods of the invention, such methods maybe used as second or third line methods of treatment for patients wheresuch patients were provided with standard therapies that failed. Forexample, cisplatin is a first line treatment for head and neck cancers.However, in certain instances, the patient may not respond to cisplatinor simply relapse after a certain period of time. In such instanceswhere the patient relapses, the cancer or neoplastic disorder can bemore difficult to treat. The present method can thus provide a second orthird line method of treatment after certain initial methodologies failor are inadequate.

Furthermore, in certain aspects of the methods of the invention, thecompounds of the invention (e.g., Formulas I-IV) may be utilized incombination with one or more other additional therapeutic agents, asnecessary. For example, such additional therapeutic agents may includebortezomib and/or dexamethasone.

Where the compounds of the invention are administered in combinationwith one or more additional therapeutic agents, the additionaltherapeutic agents may be delivered intravenously (e.g., at a dose fromabout 0.1 to 10 mg/m²) and/or orally (e.g., at a dose from about 1 to100 mg). For example, in a specific method of the invention for thetreatment of cancer, a compound selected from Formula I could beprovided to a patient in need of such treatment. After providing thecompound of the invention, the method may further include theadministration of bortezomib to the patient in combination withdexamethasone. The administration of these three compounds could beapplied in cycles over the course of days or weeks as understood by aperson having ordinary skill in the art in order to maximize theircombined effect against the cancer being treated. During the cycling ofthe compound of the invention and the additional therapeutic agents, themethod may further include the step of irradiating the patient accordingto a regimen set forth herein.

For a specific example, a method for treating cancer, such as multiplemyeloma, in patient in need thereof may include, in a first phase of themethod: providing a compound selected from Formula I to the patient oncedaily 2 to 4 times per week for about 2 weeks during a first 3 weekcycle, then providing bortezomib at a dose of about 1.3 mg/m²intravenously to the patient twice weekly for about 2 weeks during the 3week cycle; and then providing dexamethasone to the patient at a dose ofabout 20 mg orally per day of bortezomib and the day after each dose ofbortezomib.

Where the patient demonstrates or achieves a measurable clinical benefitdue to the method of the invention, the method of the invention mayfurther include a second phase that comprises: providing the compoundselected from Formula I to the patient once daily 2 to 4 times per weekfor about 2 weeks during a second 3 week cycle; then providingbortezomib at a dose of about 1.3 mg/m² intravenously to the patientonce weekly for 2 weeks during the three week cycle; and then providingdexamethasone to the patient at a dose of about 20 mg orally per day ofbortezomib and the day after each dose of bortezomib. In one embodiment,the first phase of the method may include 8 or fewer 3 week cycles.Additionally, the second phase of the method may include 8 or fewer 3week cycles. Moreover, the method of the invention may include theirradiation of the patient before, after, or during the administrationof the compound of the invention as set forth in the first phase of themethod or the second phase of the method. Such radiation may be appliedas described herein.

The following examples describe the invention in further detail. Theseexamples are provided for illustrative purposes only, and should in noway be considered as limiting the invention.

EXAMPLES Example 1: Synthesis ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide (SeeFIGS. 1 and 2)

In a first step, we prepared the intermediate methyl6-(3-methyl-1H-indole-2-carboxamido)hexanoate. Triethylamine (1.73 g,17.1 mmol) was added to a solution of 3-methyl-1H-indole-2-carboxylicacid (300 mg, 1.71 mmol) in dimethyl formamide (DMF) and the solutionwas cooled to 0° C. using an ice bath. PyBop (1.337 g, 2.57 mmol) wasadded and the solution was allowed to stir for 15 minutes after whichmethyl-6-amino hexanoate HCl (311 mg, 1.71 mmol) was added. The solutionwas allowed to stir overnight while warming to room temperature. DMF wasthen removed under reduced pressure and the remaining residue was takenup in ethyl acetate (EtOAc), and the organic solution was successivelywashed with brine and a saturated LiCl solution. The organic portion wasthen evaporated and the residue was purified via column chromatographyusing Hexanes:EtOAc to yield 413.4 mg of a tan solid (80% yield).

Characterization of methyl6-(3-methyl-1H-indole-2-carboxamido)hexanoate: ¹HNMR, Cl₃CD, 400 MHz δ:9.19 (1H, s-br), 7.61 (1H, d), 7.37 (1H, d), 7.27 (1H, t), 7.13 (1H, t),3.66 (3H, s), 3.53 (2H, m), 2.57 (3H, s), 1.69 (4H, m), 1.45 (2H, m),1.27 (2H, m). ¹³CNMR, Cl₃CD, 60 MHz, δ: 174.16, 155.07, 148.22, 128.71,124.67, 120.00, 119.83, 111.69, 51.51, 33.80, 31.55, 29.37, 26.35,24.40, 14.07.

We then prepared theN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide productfrom the hexanoate intermediate. 4.5 ml of hydroxylamine (50% solutionin H₂O) was added to a solution of methyl6-(3-methyl-1H-indole-2-carboxamido)hexanoate (413 mg, 1.37 mmol) inmethanol and the solution was heated to 60° C. overnight. The reactionwas quenched with the addition of acetone and the solvent and acetoneoxime were removed under reduced pressure. The remaining residue waspurified via column using EtOAc:MeOH to yield a tan solid (257 mg, 62%).

Characterization ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide: ¹HNMR,DMSO-d₆, 400 MHz δ: 11.32 (1H, s), 10.37 (1H, s), 8.67 (1H, s-br), 7.99(1H, s), 7.54 (1H, d), 7.34 (1H, d), 7.16 (1H, t), 7.00 (1H, t), 3.25(2H, m), 2.47 (3H, s), 1.95 (2H, m), 1.52 (4H, m), 1.30 (2H, m). ¹³CNMR,DMSO-d₆, 60 MHz, δ: 169.59, 162.33, 135.71, 128.45, 128.18, 124.02,120.01, 119.34, 113.94, 112.21, 39.14, 32.68, 29.34, 26.57, 25.35,10.12.

Example 2: Synthesis ofN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide (See FIG.1)

In a first step, we prepared the methyl8-((2-(2-methyl-1H-indol-3-yl)ethyl)amino)-8-oxooctanoate intermediate.A solution of 2-(2-methyl-1H-indol-3-yl)ethylamine (252 mg, 1.45 mmol)in dichloromethane (DCM) was added dropwise to a solution of methyl8-chloro-8-oxooctanoate (300 mg, 1.45 mmol) and solid K₂CO₃ in DCM andthe mixture was allowed to stir for 2 hours. Once the reaction wascompleted as determined by thin layer chromatography (TLC), the reactionwas quenched with the addition of 1M sulfuric acid until the pH becameslightly acidic. The solution was the extracted once with water followedby brine after which the organic layers were dried over sodium sulfate.The solvent was then removed via vacuum and the residue was purifiedover a column using 0-50% EtOAc in Hexanes to yield 338.7 mg (68%).

Characterization of methyl8-((2-(2-methyl-1H-indol-3-yl)ethyl)amino)-8-oxooctanoate: ¹HNMR, Cl₃CD,400 MHz δ: 8.22 (1H, s-br), 7.47 (1H, d), 7.26 (1H, d), 7.08 (2H, m),5.63 (1H, s-br), 3.66 (3H, s), 3.50 (2H, q), 2.90 (2H, t), 2.36 (3H, s),2.27 (2H, t), 2.06 (2H, t), 1.56 (4H, m), 1.26 (4H, m). ¹³CNMR, Cl₃CD,60 MHz, δ: 174.28, 173.08, 135.35, 132.03, 128.61, 121.06, 119.28,117.70, 110.39, 108.31, 51.46, 39.91, 36.60, 33.94, 28.80, 28.75, 25.41,24.68, 24.12, 11.58.

We then prepared theN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide productfrom the octanoate intermediate. 3 ml of hydroxylamine (50% solution inH₂O) was added to a solution of methyl8-((2-(2-methyl-1H-indol-3-yl)ethyl)amino)-8-oxooctanoate (338.7 mg,0.98 mmol) in methanol and the solution was heated to 60° C. overnight.The reaction was quenched with the addition of acetone and the solventand acetone oxime were removed under reduced pressure. The remainingresidue was purified via column using EtOAc:MeOH to yield a white solid(182 mg, 54%).

Characterization ofN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide: ¹HNMR,DMSO-d₆, 400 MHz δ: 10.68 (1H, s), 10.35 (1H, s), 8.66 (1H, s-br), 7.83(1H, s), 7.41 (1H, d), 7.22 (1H, d), 6.94 (2H, m), 3.19 (2H, q), 2.74(2H, t), 2.30 (3H, s), 2.03 (2H, t), 1.94 (2H, t), 1.47 (4H, m), 1.22(4H, m). ¹³CNMR, DMSO-d₆, 60 MHz, δ: 172.41, 166.64, 135.65, 132.46,128.79, 120.30, 118.48, 117.73, 110.78, 108.12, 40.06, 35.91, 32.71,28.91, 28.86, 25.59, 25.48, 24.72, 11.63.

Example 3: Synthesis of(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide (SeeFIG. 2)

In a first step, we prepared the intermediate(S)-1-tosylindoline-2-carboxylic acid. p-Toluene sulfonyl chloride (1.17g, 6.13 mmol) was added to a stirred solution of(s)-(−)-indoline-2-carboxylic acid (1 g, 6.13 mmol) with K₂CO₃ in DCM.The mixture was allowed to stir overnight and was quenched with theaddition of 1M sulfuric acid. The solution was then extracted with 1Msulfuric acid and the organic layers were dried over sodium sulfate.After filtration, the solvent was evaporated and the residue waspurified over a column using 0-10% MeOH in DCM yielding 852 mg of asticky oil (44%).

Characterization of (S)-1-tosylindoline-2-carboxylic acid: ¹HNMR,DMSO-d₆, 400 MHz δ: 7.64 (2H, d), 7.43 (1H, d), 7.31 (2H, d), 7.18 (1H,t), 7.07 (1H, d), 6.97 (1H, t), 4.70 (1H, dd), 3.03 (1H, dd), 2.87 (1H,dd), 2.30 (3H, s).

We then prepared a(S)-methyl 6-(1-tosylindoline-2-carboxamido)hexanoateintermediate from the carboxylic acid. Triethylamine (1.73 g, 17.1 mmol)was added to a solution of (S)-1-tosylindoline-2-carboxylic acid (524.8mg, 1.65 mmol) in DMF and the solution was cooled to 0° C. using an icebath. PyBop (1.29 g, 2.48 mmol) was added to the solution and allowed tostir for 15 minutes after which methyl-6-amino hexanoate HCl (300 mg,1.65 mmol) was added. The solution was allowed to stir overnight whilewarming to room temperature. DMF was then removed under reduced pressureand the remaining residue was taken up in EtOAc, and the organic portionwas washed successively with brine and a saturated LiCl solution. Theorganic layer was then evaporated and the residue was purified viacolumn chromatography using Hexanes:EtOAc to yield 498.8 mg of an oil(68% yield).

Characterization of (S)-methyl6-(1-tosylindoline-2-carboxamido)hexanoate: ¹HNMR, Cl₃CD, 400 MHz δ:9.19 (1H, s), 7.64 (2H, d), 7.43 (1H, d), 7.31 (2H, d), 7.18 (1H, t),7.07 (1H, d), 6.97 (1H, t), 4.70 (1H, dd), 3.66 (3H, s), 3.09 (2H, m),3.03 (1H, dd), 2.87 (1H, dd), 2.30 (3H, s), 1.92 (2H, t), 1.47 (2H, m),1.40 (2H, m), 1.23 (2H, m).

We then preparedthe(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamideproduct from hexanoate intermediate. 4.5 ml of hydroxylamine (50%solution in H₂O) was added to a solution of (S)-methyl6-(1-tosylindoline-2-carboxamido)hexanoate (498 mg, 1.12 mmol) inmethanol and the solution was heated to 60° C. overnight. The reactionwas quenched with the addition of acetone and the solvent and acetoneoxime were removed under reduced pressure. The remaining residue waspurified via column using EtOAc:MeOH to yield a clear oil (219.5 mg,44%).

Characterization of(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide:¹HNMR, DMSO-d₆, 400 MHz δ: 10.33 (1H, s), 8.64 (1H, s-br), 8.07 (1H, t),7.64 (2H, d), 7.43 (1H, d), 7.31 (2H, d), 7.18 (1H, t), 7.07 (1H, d),6.97 (1H, t), 4.70 (1H, dd), 3.09 (2H, m), 3.03 (1H, dd), 2.87 (1H, dd),2.30 (3H, s), 1.92 (2H, t), 1.47 (2H, m), 1.40 (2H, m), 1.23 (2H, m).¹³CNMR, DMSO-d₆, 60 MHz, δ: 170.56, 169.55, 144.40, 141.17, 133.85,130.92, 129.82, 127.56, 127.19, 125.11, 124.34, 115.35, 62.80, 38.60,36.43, 33.06, 26.57, 25.35, 20.94, 14.05.

Example 4: Synthesis of(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(See FIG. 2)

In a first step, we prepared(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)indoline-2-carboxylicacid. Dansyl chloride (1.65 g, 6.13 mmol) was added to a stirredsolution of (s)-(−)-indoline-2-carboxylic acid (1 g, 6.13 mmol) withK₂CO₃ in DCM. The mixture was allowed to stir overnight and was quenchedwith the addition of 1M sulfuric acid. The solution was then extractedwith 1M sulfuric acid and the organic layers were dried over sodiumsulfate. After filtration, the solvent was evaporated and the residuewas purified over a column using 0-10% MeOH in DCM yielding 1.28 g of ayellow solid (53%).

Characterization of prepared(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)indoline-2-carboxylicacid: ¹HNMR, DMSO-d₆, 400 MHz δ: 13.23 (1H, s-br), 8.44 (1H, d), 8.19(1H, d), 8.16 (1H, d), 7.56 (2H, m), 7.21 (1H, d), 7.13 (3H, m), 6.92(1H, t), 5.06 (1H, dd), 3.36 (1H, dd), 3.07 (1H, dd), 2.77 (6H, s).

We then prepared the (S)-methyl6-(1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)indoline-2-carboxamido)hexanoateintermediate from the carboxylic acid. Triethylamine (1.27 g, 12.6 mmol)was added to a solution of(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)indoline-2-carboxylicacid (500 mg, 1.26 mmol) in DMF and the solution was cooled to 0° C.using an ice bath. PyBop (984.4 mg, 1.85 mmol) was added to the solutionand allowed to stir for 15 minutes after which methyl-6-amino hexanoateHCl (228.9 mg, 1.26 mmol) was added. The solution was allowed to stirovernight while warming to room temperature. DMF was then removed underreduced pressure and the remaining residue was taken up in EtOAc, theorganic portion was washed successively with brine and a saturated LiClsolution. The organic layer was then evaporated and the residue waspurified via column chromatography using Hexanes:EtOAc to yield 475 mgof a yellow oil (72% yield).

Characterization of (S)-methyl6-(1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)indoline-2-carboxamido)hexanoate:¹HNMR, Cl₃CD, 400 MHz δ: 9.19 (1H, s) 8.44 (1H, d), 8.19 (1H, d), 8.16(1H, d), 7.56 (2H, m), 7.21 (1H, d), 7.13 (3H, m), 6.92 (1H, t), 5.06(1H, dd), 3.66 (3H, s), 3.36 (1H, dd), 3.09 (2H, m), 3.07 (1H, dd), 2.77(6H, s), 1.92 (2H, t), 1.47 (2H, m), 1.40 (2H, m), 1.23 (2H, m).

We then prepared the(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamideproduct from the hexanoate. 4.5 ml of hydroxylamine (50% solution inH₂O) was added to a solution of (S)-methyl6-(1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)indoline-2-carboxamido)hexanoate(475 mg, 0.905 mmol) in methanol and the solution was heated to 60° C.overnight. The reaction was quenched with the addition of acetone andthe solvent and acetone oxime were removed under reduced pressure. Theremaining residue was purified via column using EtOAc:MeOH to yield ayellow oil (270 mg, 52%).

Characterization of (S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide: ¹HNMR,DMSO-d₆, 400 MHz δ: 10.29 (1H, s), 8.64 (1H, s-br), 8.45 (1H, d), 8.18(1H, d), 8.12 (1H, d), 8.00 (1H, s), 7.59 (1H, t), 7.47 (1H, t), 7.27(1H, t), 7.20 (1H, d), 7.14 (1H, t), 7.08 (1H, t), 6.96 (1H, t), 4.88(1H, dd), 3.09 (2H, m), 3.03 (1H, dd), 2.87 (1H, dd), 2.77 (6H, s), 1.92(2H, t), 1.47 (2H, m), 1.40 (2H, m), 1.23 (2H, m). ¹³CNMR, DMSO-d₆, 60MHz, δ: 174.44, 170.11, 151.34, 144.40, 143.37, 133.02, 129.82, 128.34,127.96, 127.19, 126.35, 125.11, 124.91, 124.34, 123.77, 119.93, 117.61,115.35, 62.80, 46.23, 38.60, 36.43, 33.06, 26.57, 25.35, 14.05.

Example 5: Activity ofN-(6-(carboxy)-6-oxohexyl)-1H-indole-2-carboxamide (SP-1-105) as anActivator of ATM (See FIGS. 3A and 3B)

Several assays were utilized to determine the ability of the compoundsof the invention to activate ATM over a period of time. ATM activationmay be demonstrated by phosphorylation of ATM (i.e., phosphor-ATM) inthe MCF-7 cell line. The fold change in phospho-ATM was measured over aperiod of time. Accordingly, the increase in phospho-ATM indicatesactivation of ATM by the compounds of the invention.

In certain examples, the activity of the compounds of the invention wascompared to additional modulators of ATM, such as KU55399 (a specificATM inhibitor); dimethylsulfoxide (DMSO) (a solvent);3,3′-diindolylmethane (DIM) (an ATM activator); and irradiation(irradiation may result in the generation of phospho-ATM due to DNAdamage). DIM was not used as a comparative compound in Example 5.

Materials: MCF7 Cells; Complete RPMI media: RPMI, 10% Fetal Bovine Serum(FBS), 5% L-glutamine, 5% Pen/Strep; R&D Systems Human P-ATM (S1981)DuoSet IC ELISA Assay Kit; Plate sealers; Normal mouse serum (heatinactivated 56 C for 30 min); 96-well ELISA microtiter plate; 450 nmPlate Reader; 60 mm dishes; Disposable pipettes; Pipet-aid; Pipettetips; Micro-pipetter; Cell scraper; Distilled water; Pierce NE-PERNuclear and Cytoplasmic Extraction Reagent; Pierce Protease andPhosphotase Inhibitor Mini Tablets; KU-55933; DIM (not used in Example5); Dimethyl sulfoxide (DMSO); Phosphate buffered saline (PBS); andSterile micro-tubes and conical tubes; Hemocytometer; Microscope;Micro-centrifuge.

MCF7 Cells were grown in Complete RPMI media and 10⁶ cells were seededinto 60 mm dishes. Dishes were then divided into duplicate treatmentsthe following day. Control samples were treated with 3 ml RPMI media for1 hr. Vehicle samples were treated with 3 ml RPMI media plus 0.1% DMSOfor 1 hr. Negative control samples were treated with 10 μM KU-55933 in 3ml RPMI media plus 0.1% DMSO for 1 Hr. Positive control samples weretreated with 0.5 μM DIM in 3 ml RPMI media plus 0.1% DMSO for 30 min.Test Samples were treated with 1 μM of the test compounds (e.g.N-(6-(hydroxyamino)-6-oxohexyl)-1H-indole-2-carboxamide in Example 5) in3 ml RPMI media plus 0.1% DMSO for 30 min; 1 hr; 2 hrs; 4 hrs; and 6hrs. At their designated time, samples were harvested on ice using acell scraper and two washes of 1 ml PBS and kept as a cell pellet on dryice until all were collected. After all samples were collected they werethawed on ice and separated into cytosolic and nuclear fractions usingthe Pierce NE-PER Nuclear and Cytoplasmic Extraction Reagent kit withadded Pierce protease and phosphotase inhibitor tablet.

After recovery of the separate fractions, the process further includedwashing cells by suspending the cell pellet with PBS. 1-10×106 cellswere transferred to a 1.5 mL microcentrifuge tube and a pellet wasformed by centrifugation at 500×g for 2-3 minutes. 100 μL of ice-coldCER I was added to the cell pellet. The tube was vortexed vigorously onthe highest setting for 15 seconds to fully suspend the cell pellet andincubate the tube on ice for 10 minutes. 5.5 μL of ice-cold CER II wasadded to the tube. The tube was vortexed for 5 seconds on the highestsetting and the tube was incubated on ice for 1 minute. The tube wasvortexed for 5 seconds on the highest setting and centrifuged for 5minutes at maximum speed in a microcentrifuge (˜16,000×g). Thesupernatants were used as the cytoplasmic fraction and were discarded.The insoluble (pellet) fraction, which contains nuclei, was thensuspended in 50 μL of ice-cold NER. The fraction was vortexed on thehighest setting for 15 seconds and the samples were placed on ice andvortexing continued for 15 seconds every 10 minutes, for a total of 40minutes. The tubes were centrifuged at maximum speed (˜16,000×g) for 10minutes. The nuclear fraction supernatants were placed on ice until usedin the R&D Systems Human Phospho-ATM (S1981) DuoSet IC ELISA Assay Kit.

Turning to the ELISA kit, the following steps were used in accordancewith the instructions.

Plate Preparation. The Capture Antibody was diluted to a workingconcentration 10.0 μg/ml in PBS without carrier protein. Immediatelycoat a 96-well microplate with 100 μL per well of the diluted CaptureAntibody. The plate was sealed and incubated overnight at roomtemperature. Each well was aspirated and washed with Wash Buffer, withthe process repeated two times for a total of 3 washes. The wells werewashed by filling each well with Wash Buffer (400 μL). After the lastwash, any remaining Wash Buffer was removed by aspirating. The plateswere blocked by adding 300 μL of Block Buffer to each well. The plateswere then incubated at room temperature for 2 hours. The aspiration/washcycle was repeated. The plates were then ready for sample addition.

Proceeding with sample addition, a Phospho-ATM Standard was prepared byreconstituting with 500 μL of IC Diluent #4. A seven point curve wasdeveloped using 2-fold serial dilutions in IC Diluent #4 and a highstandard of 200 ng/mL was used to make the standard curve. 100 μL ofstandard was added per well of plate. 50 μL of sample mixed with 50 μLof IC diluents #4 was added per well of the plate. The IC Diluent #4 wasused as the blank. A plate sealer was used to cover the plate and theplate was then incubated for 2 hours at room temperature. Theaspiration/wash cycle was repeated as described above for PlatePreparation. Immediately before use, the Detection Antibody was dilutedto 200 ng/ml in IC Diluent #1 that contained 2% heat-inactivated normalmouse serum. Only as much Detection Antibody was prepared as required torun each assay. The diluted Detection Antibody was allowed to sit 2hours before use. 100 μL of the diluted Detection Antibody was added toeach well. The plate was covered with a new plate sealer and incubated 2hours at room temperature. The aspiration/wash cycle was repeated asdescribed above for Plate Preparation. Immediately before use,Streptavidin-HRP was diluted to the working concentration specified onthe vial label using IC Diluent #1. 100 μL of the dilutedStreptavidin-HRP was added to each well. The plates were incubated for20 minutes at room temperature. Placing the plate in direct light wasavoided. The aspiration/wash step was repeated as described above. 100μL of Substrate Solution was added to each well. The plates wereincubated for 20 minutes at room temperature. Placing the plate indirect light was avoided. 50 μL of Stop Solution was then added to eachwell. The plate was gently tapped to ensure thorough mixing. The opticaldensity of each well was determined immediately, using a microplatereader set to 450 nm. Where wavelength correction was available, it wasset to 540 nm or 570 nm. Experimental samples were compared to controlsand the standard curve.

After the indicated treatment, whole cell lysates were assayed forprotein phosphatise 2A (PP2A) enzymatic activity by using a PP2AImmunoprecipitation Phosphatase Assay Kit, according to themanufacturer's instructions.

Materials: MCF7 Cells; Complete RPMI media: RPMI, 10% Fetal Bovine Serum(FBS), 5% L-glutamine, 5% Pen/Strep; Millipore PP2A ImmunoprecipitationPhosphatase Assay Kit; 96-well EISA microtiter plate; 650 nm PlateReader; 60 mm dishes; Disposable pipettes; Pipet-aid; Pipette tips;Micro-pipetter; Cell scraper; Distilled water; Phosphatase extractionbuffer: 20 mM imidazole-HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0, with 10 μg/mLeach of aprotinin, leupeptin, antipain, soybean trypsin inhibitor, 1 mMbenzamidine, and 1 mM PMSF; Okadaic acid (OAC); Dimethyl sulfoxide(DMSO); Sterile micro-tubes and conical tubes; Hemocytometer;Microscope; and Micro-centrifuge.

MCF7 Cells were grown in Complete RPMI media and 10⁶ cells were seededinto 60 mm dishes. Dishes were then divided into duplicate treatmentsthe following day. Control samples were treated with 3 mL RPMI media for1 hr. Vehicle samples were treated with 3 ml RPMI media plus 0.1% DMSOfor 1 hr. Negative control samples were treated with 5 nM OAC in 3 mLRPMI media plus 0.1% DMSO for 30 min. Test Samples were treated with 1μM of each Test Compound (e.g.,N-(6-(hydroxyamino)-6-oxohexyl)-1H-indole-2-carboxamide in Example 5) in3 ml RPMI media plus 0.1% DMSO for 30 min; 1 hr; 2 hrs; 4 hrs; and 6hrs.

At their designated time, samples were harvested on ice using a cellscraper and 0.2 ml of phosphatase extraction buffer and kept on dry iceuntil all were collected. After all samples were collected they werethawed on ice, sonicated for ten seconds and centrifuge at 2000×g for 5minutes. The supernatants were then used for phosphatase activityassays. Prior to performing the phosphatase activity assays, allsolutions were tested for phosphate contamination. To determinephosphate contamination, Malachite Green Detection Solution was used.Malachite Green Phosphate Detection Solution was prepared by adding 10μL of Solution B to each 1 ml of Solution A. Only enough reagentrequired for use that day was made and kept at room temperature. 100 μLof mixed solution AB was used per assay well to 25 μL of water orbuffer, or 5 μL of the experimental sample. Samples or solutionscontaminated with phosphate would turn green. Contaminating phosphatecould be removed by desalting column or dialysis.

Next a Phosphate Standard Curve was made. This was done by diluting 125μL of Catalog #20-103, Phosphate Standard (Solution C) with 1125 μL ofdistilled water to make 0.1 μM working solution. The standard curve thatwas made ranged between 200 and 2000 pmoles per well. 25 μL of eachphosphate standard dilution was transferred to the wells of themicrotiter plate. Distilled water was used as a blank. 100 μL ofMalachite Green Solution was added to wells of standards. Wells weremixed carefully without creating bubbles (bubbles interfere withmeasurement of absorbance). Color was allowed to develop for 15 minutesat room temperature. Absorbance was measured at a wavelength of 650 nmin a microtiter plate reader. Absorbance of blank solution wassubtracted from standards and absorbance vs. pmoles phosphate wasplotted to form the standard curve.

The experimental samples were assayed using threonine phosphopeptide asthe substrate. 1 mg Threonine Phosphopeptide (Catalog #12-219) wasdissolved in 1.10 mL of distilled water to prepare a 1 mM solution. Theexperimental samples were tested by transferring their supernatants intoclean micro-tubes.

The following were then added to each sample tube:

1) 4 μg of Anti-PP2A, C subunit, clone 1D6 (Catalog #05-421).

2) 25 μL Protein A agarose beads slurry (Catalog #16-125D).

3) pNPPSer/Thr Assay Buffer (Catalog #20-179) or TBS to make totalvolume 500 μL. Sample tubes were incubated for 2 hrs at 4° C. withconstant rocking. Sample Beads were washed 3 times with 700 μL TBS,followed by one wash with 500 μL Ser/Thr Assay Buffer.

4) 60 μL of 1 mM threonine phosphopeptide was added (final reactionconcentration was 750 μM).

5) 20 μL of Ser/Thr Assay Buffer was added. Tubes were incubated for 10minutes at 30° C. in a shaking incubator. Tubes were then centrifugedbriefly and 25 μL of each sample was transferred into the microtiterplate.

6) 100 μL of Malachite Green Phosphate Detection Solution (Page two,Section A) was added and color was developed for 15 minutes at roomtemperature. The microtiter plate was read at 650 nm and absorbancevalues of experimental samples were compared to controls and standardcurve.

The results of these studies are set forth in FIGS. 3A and 3B where aconcentration of 1 μMN-(6-(hydroxyamino)-6-oxohexyl)-1H-indole-2-carboxamide was used over atime course of 6 hours. The compoundN-(6-(hydroxyamino)-6-oxohexyl)-1H-indole-2-carboxamide was compared toKU55399, DMSO, and radiation (irradiation of the MCF7 cells at 6 Gy).

Example 6: Activity ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161) as an Activator of ATM (See FIGS. 4A and 4B)

The activity ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide as anactivator of ATM in MCF7 cells was determined as set forth in Example 5.Moreover, the activity ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide wascompared to DIM.

The results of these studies are set forth in FIGS. 4A and 4B where aconcentration of 1 μMN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide wasused over a time course of 6 hours. The compoundN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide wascompared to KU55399, DMSO, DIM, and radiation (irradiation of the MCF7cells at 6 Gy).

Example 7: Activity ofN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide (SP-1-163)as an Activator of ATM (FIGS. 5A and 5B)

The activity ofN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide as anactivator of ATM in MCF7 cells was determined as set forth in Example 5.Moreover, the activity ofN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide wascompared to DIM.

The results of these studies are set forth in FIGS. 5A and 5B where aconcentration of 1 μMN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide was usedover a time course of 6 hours. The compoundN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide wascompared to KU55399, DMSO, DIM, and radiation (irradiation of the MCF7cells at 6 Gy).

Example 8: Activity of(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide(SP-1-169) as an Activator of ATM (FIGS. 6A and 6B)

The activity of(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide as anactivator of ATM in MCF7 cells was determined as set forth in Example 5.Moreover, the activity of(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide wascompared to DIM.

The results of these studies are set forth in FIGS. 6A and 6B where aconcentration of 1 μM(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide wasused over a time course of 6 hours. The compound(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide wascompared to KU55399, DMSO, DIM, and radiation (irradiation of the MCF7cells at 6 Gy).

Example 9: Activity of(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(SP-1-171) as an Activator of ATM (FIGS. 7A and 7B)

The activity of(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamideas an activator of ATM in MCF7 cells was determined as set forth inExample 5. Moreover, the activity of(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamidewas compared to DIM.

The results of these studies are set forth in FIGS. 7A and 7B where aconcentration of 1 μM(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamidewas used over a time course of 6 hours. The compound(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamidewas compared to KU55399, DMSO, DIM, and radiation (irradiation of theMCF7 cells at 6 Gy).

Example 10: Activity of the Compounds of the Invention as HDACInhibitors (See FIGS. 8A and 8B)

Certain compounds of the invention were tested in a pan-HDAC assay todetermine the ability of such compounds to inhibit HDAC protein.Specifically, the compounds tested included:N-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161) (FIG. 8A);N¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide (SP-1-163)(FIG. 8A);(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide(SP-1-169) (FIG. 8B); and(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(SP-1-171) (FIG. 8B).

The Pan HDAC assay was performed as follows:

Materials: Enzo Fluor-de-Lys HDAC fluorometric activity assay kit; 360nm excitation 460 nm emission Plate Reader; Disposable pipettes;Pipet-aid; Pipette tips; Micro-pipetter; DMSO; Sterile micro-tubes andconical tubes; and Micro-centrifuge.

Test compounds and control preparation: All test compounds' stocksolutions were made in 100% DMSO. Test compound dilutions were made inHDAC Assay buffer with a final concentration of DMSO less than or equalto 1%. Vehicle control was HDAC Assay buffer with 1% DMSO.

The reagents for the assay were prepared as follows:

1. All kit components were defrosted and these, and all dilutionsdescribed below, were kept on ice until use. All undiluted kitcomponents are stable for several hours on ice.

2. A sufficient amount of HeLa Nuclear Extract (BML-KI140) or other HDACsource diluted in Assay Buffer (BML-KI143) was prepared to provide forthe assays that were performed (# of wells×15 μl). A 30-fold dilution ofthe HeLa Extract means that 15 μL contains 0.5 μl of the undilutedExtract, an appropriate amount to use per well.

3. Dilution(s) of Trichostatin A and/or Test Inhibitors were prepared inAssay Buffer (BML-KI143). Since 10 μl were used per well, and since thefinal volume of the HDAC reaction was 50 μl, these inhibitor dilutionswere 5× their final concentration.

4. Dilution(s) of the Fluor de Lys® Substrate (BML-KI104; 50 mM) wereprepared in Assay Buffer (BML-KI143) that were 2× the desired finalconcentration(s). For inhibitor screening, substrate concentrations ator below the Km are recommended. Twenty-five μL were used per well.Initial dilutions of 25-fold or greater in Assay Buffer (2.0 mM or less)yielded stable solutions (see NOTE on freezing and thawing below). Rapidmixing and dilution into room temperature buffer helped to preventprecipitation at high substrate concentration. NOTE: Freezing/thawing ofFluor de Lys® Substrate solutions in Assay Buffer may causeprecipitation of the Substrate. Dilute only amount necessary for oneday's experiment.

5. Shortly before use (<30 min.), sufficient Fluor de Lys® Developer wasprepared for the assays to be performed (50 μl per well). First, theFluor de Lys® Developer Concentrate was diluted 20-fold (e.g. 50 μl plus950 μl Assay Buffer) in cold Assay Buffer (BML-KI143). Second, the 0.2mM Trichostatin A (BML-GR309-9090) was diluted 100-fold in the 1×Developer just prepared (e.g. 10μl in 1 ml; final Trichostatin Aconcentration in the 1× Developer=2 μM; final concentration afteraddition to HDAC/Substrate reaction=1 μM). The addition of TrichostatinA to the Developer insures that HDAC activity stops when the Developeris added. The Developer was kept on ice until use.

Assay Procedure:

1. Assay buffer, diluted trichostatin A or test inhibitor was added toappropriate wells of the microtiter plate.

2. Diluted HeLa extract or other HDAC sample was added to all wellsexcept those that are to be “No Enzyme Controls.”

3. Diluted Fluor de Lys® Substrate and the samples in the microtiterplate were allowed to equilibrate to assay temperature (e.g. 25 or 37°C.).

4. HDAC reactions were initiated by adding diluted substrate (25 μl) toeach well and mixing thoroughly.

5. HDAC reactions were allowed to proceed for desired length of time andthen stop them by addition of Fluor de Lys® Developer (50 μl). Theplates were incubated at room temperature (25° C.) for 10-15 min. Signalis stable for at least 30 min. beyond this time.

6. Samples were read in a microtiter-plate reading fluorimeter capableof excitation at a wavelength in the range 350-380 nm and detection ofemitted light in the range 440-460 nm.

A shown in FIGS. 8A and 8B, the tested compounds demonstrated inhibitoractivity in the Pan-HDAC assay. For example, as shown in FIG. 8A,N-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide andN¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide (SP-1-163)demonstrated an average EC₅₀ of 0.02971 μM and 0.1117 μM, respectively.Moreover, as shown in FIG. 8B,(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide(SP-1-169) and(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(SP-1-171) demonstrated an average EC₅₀ of 0.2649 μM and 0.02533 μM,respectively.

Example 11: Cytotoxicity Testing of the Compounds of the Invention inBreast Cancer Cells (MCF7 Cells) and Normal Breast Cells (184A1 Cells)(See FIG. 9)

Certain compounds of the invention were tested in both an MTTcytotoxicity assay and clonogenic cytotoxicity assay against breastcancer cells (MCF7 cells) and normal breast tissue cells (184A1 cells)to determine the cytotoxic effect of such cells as a function ofconcentration.

Specifically, the compounds tested included:N-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161); N¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)ethyl)octanediamide(SP-1-163);(S)—N-(6-(hydroxyamino)-6-oxohexyl)-1-tosylindoline-2-carboxamide(SP-1-169); and(S)-1-((5-(dimethylamino)naphthalene-1-yl)sulfonyl)-N-(6-(hydroxyamino)-6-oxoheyl)indoline-carboxamide(SP-1-171).

As demonstrated in FIG. 9, SP-1-161 was the most cytotoxic having IC₅₀sof 0.416 and 0.276 μM in the MTT assay and clonogenic assay,respectively.

Example 12: Comparison of the Cytoxic Activity ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161) with DIM (FIGS. 11A and 11B)

N-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide(SP-1-161) was tested against DIM in a clonogenic survival study withhealthy breast epithelial cells (184A1 cells) (FIG. 11A) and breastcancer cells (MCF7 cells) (FIG. 11B). After treatment with thecompounds, the cells were irradiated.

Logarithmically growing cells were seeded into T-25 flasks at variousdensities to yield approximately 50-100 colonies/flask. After incubationfor 24 h at 37° C., cells were treated or not with the compound (IC50)for 24 h followed by sham or irradiation at room temperature using aMark-30 irradiator with a 137-Cs source at a fixed dose rate of 2.27Gy/min. After 10-14 days, cells were fixed and stained with crystalviolet, and colonies of more than 50 cells were counted. The survivingfractions of the treated cells were normalized to the platingefficiencies of untreated controls. Radiation survival curves werefitted by computer to the single-hit, multitarget and thelinear-quadratic models.

As demonstrated in FIG. 11B, SP-1-161 outperformed DIM in treatingbreast cancer cells (MCF7) when combined with radiation. Moreover, FIG.11A demonstrates that the surviving population of healthy cells treatedwith SP-1-161 was comparable to that of DIM, indicating that thatSP-1-161 provided protective activity comparable to DIM.

Example 13: Comparison of the Effect of Indole Methylation on ATMActivation (FIG. 12)

Several compounds of the invention include alkylated indole moieties. Inan effort to better understand the compounds of the invention, and theirbiological activity, we developed a study to measure the ATM activatingproperties of substituted and unsubstituted indoles.

Without being restricted to any one theory of the invention, it isbelieved that the compounds of the invention derive ATM activationactivity from one or more indole moieties as described in Formulas I-IV.See, for example, SP-1-161 and SP-1-163. To test this understanding,certain indole species were tested for their effects on ATMphosphorylation (i.e., an indicator of ATM activation) in the absence ofa hydroxamic acid moiety. Specifically, this study examined a homologousseries of indoles that included the unsubstituted indole, methylindole,and dimethylindole.

The activity of indole, 3-methyl-indole, and 2,3-dimethyl indole wasdetermined as set forth in Example 5 (FIG. 12).

The results of these studies are set forth in FIG. 12 where aconcentration of 1 μM indole, 3-methyl-indole, and 2,3-dimethyl indolewas used over a time course of 6 hours. The methylated indoles werecompared to DMSO (irradiation of the MCF7 cells at 6 Gy).

As shown in FIG. 12, ATM-activation, as determined by the fold change inphospho-ATM, occurred with all indole species as compared to DMSO.Surprisingly, there was marked difference between indole and the testedmethylated indoles. Specifically, as methylation increased, there was amarked increase in the level of ATM phosphorylation. Indeed,2,3,-dimethylindole showed a significant increase in ATM activationbetween 1 and 2 hours pos-radiation exposure as compared to indole and3-methyl indole.

This study indicates that indole species may provide for direct ATMactivation where such tested indole species increased the level of ATMphosphorylation as compared to a control. Moreover, the level ofmethylation about the indole ring correlated to an increased level ofATM activation, as compared to unsubstituted indole.

Extrapolating this data to the compounds of the invention, we may betterunderstand the exceptional activities ofN-(6-(hydroxyamino)-6-oxohexyl)-3-methyl-1H-indole-2-carboxamide (i.e.,SP-1-161) and N¹-hydroxy-N⁶-(2-(2-methyl-1H-indol-3-yl)octanediamide(i.e., SP-1-163). Indeed, both SP-1-161 and SP-1-163 both includedisubstituted indoles and display exemplary ATM activation activity asdual function agents of the invention.

A number of patent and non-patent publications are cited herein in orderto describe the state of the art to which this invention pertains. Theentire disclosure of each of these publications is incorporated byreference herein.

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing disclosure. The presentinvention is, therefore, not limited to the particular embodimentsdescribed and/or exemplified, but is capable of considerable variationand modification without departure from the scope of the appendedclaims.

Moreover, as used herein, the term “about” means that dimensions, sizes,formulations, parameters, shapes and other quantities andcharacteristics are not and need not be exact, but may be approximateand/or larger or smaller, as desired, reflecting tolerances, conversionfactors, rounding off, measurement error and the like, and other factorsknown to those of skill in the art. In general, a dimension, size,formulation, parameter, shape or other quantity or characteristic is“about” or “approximate” whether or not expressly stated to be such. Itis noted that embodiments of very different sizes, shapes and dimensionsmay employ the described arrangements.

Furthermore, the transitional terms “comprising”, “consistingessentially of” and “consisting of”, when used in the appended claims,in original and amended form, define the claim scope with respect towhat unrecited additional claim elements or steps, if any, are excludedfrom the scope of the claim(s). The term “comprising” is intended to beinclusive or open-ended and does not exclude any additional, unrecitedelement, method, step or material. The term “consisting of” excludes anyelement, step or material other than those specified in the claim and,in the latter instance, impurities ordinary associated with thespecified material(s). The term “consisting essentially of” limits thescope of a claim to the specified elements, steps or material(s) andthose that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. All compounds, compositions,and methods described herein that embody the present invention can, inalternate embodiments, be more specifically defined by any of thetransitional terms “comprising,” “consisting essentially of,” and“consisting of.”

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What is claimed is:
 1. A method of treating a disease in a patient inneed thereof, wherein said treatment comprises administering atherapeutically effective amount of at least one compound of the formula(II):

wherein R¹¹, R¹³, R ¹⁴, R¹⁶ are independently selected from the groupconsisting of H, hydroxy, halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted cycloalkyl, optionally substituted aryl,optionally substituted heterocycle, optionally substituted heteroaryl,optionally substituted amino, optionally substituted alkoxy, optionallysubstituted carboxy, optionally substituted carbalkoxy, optionallysubstituted carboxamido, substituted sulfonyl, substituted sulfinyl,optionally substituted monoalkylaminosulfinyl, optionally substituteddialkylaminosulfinyl, optionally substituted monoalkylaminosufonyl,optionally substituted dialkylaminosulfonyl, optionally substitutedalkyl sulfonylamino, optionally substituted hydroxysulfonyloxy,optionally substituted alkoxysulfonyloxy, optionally substituted alkylsulfonyloxy, optionally substituted hydroxysulfonyl, optionallysubstituted alkoxysulfonyl, optionally substituted alkyl sulfonylalkyl,optionally substituted monoalkylaminosulfonylalkyl, optionallysubstituted dialkylaminosulfonylalkyl, optionally substitutedmonoalkylaminosulfinylalkyl, and optionally substituteddialkylaminosulfinylalkyl; X is selected from the group consisting of:

R¹² and R¹⁵ are each independently selected from the group consisting ofH, optionally substituted alkyl, substituted sulfinyl, and substitutedsulfonyl; R¹⁷ is selected from the group consisting of H and optionallysubstituted alkyl; n is an integer from 3 to 10; the dashed line mayindicate the presence of a single bond or a double bond as allowed; orthe pharmaceutically acceptable salts thereof.
 2. A method of treating adisease in a patient in need thereof, wherein said treatment comprisesadministering a therapeutically effective amount of a compound selectedfrom the group consisting of:

and the pharmaceutically acceptable salts thereof, wherein the compoundinhibits histone deacetylase (HDAC) and activates ataxia telangiectasiamutated (ATM).
 3. The method of claim 1 or claim 2, wherein the compoundis administered in dosage unit form.
 4. The method of claim 3, whereinthe dosage unit includes a physiologically compatible carrier medium. 5.The method of claim 1 or claim 2, wherein the disease is selected fromthe group consisting of cancer, an immunological disorder, and aneurological disorder.
 6. The method of claim 5, wherein said cancer isselected from the group consisting of gastric cancer, prostate cancer,colon cancer, breast cancer, Non-Hodgkin's lymphoma, ovarian cancer,sarcoma, lung cancer, leukemia, myeloma, testicular cancer, cervicalcancer, pancreatic cancer, head and neck cancer, rectal cancer, andbrain cancer.
 7. The method of claim 6, further including the step ofadministering to said patient an amount of radiotherapy configured totreat said cancer.
 8. The method of claim 1 or claim 2, wherein themethod is a second line method of treatment for the patient andadministration of the compound occurs after performance of a first linetherapy on the patient that failed to treat the disease.
 9. The methodof claim 1 or claim 2, wherein the method is a third line method oftreatment for the patient and administration of the compound occursafter performance of a second line therapy on the patient that failed totreat the disease.
 10. The method of claim 7, wherein cancerous cellsare sensitized to radiotherapy by inhibiting histone deacetylase (HDAC)and non-cancerous cells are protected from radiotherapy by activatingataxia telangiectasia mutated (ATM).